Scientia Agricultura Sinica

Study on Resistance Mechanism to Blast Mediated by P450 Gene Oscyp71Z2 in Rice

LI Wen-Qi-1, WANG Fang-Quan-1, WANG Jun-1, FAN Fang-Jun-1, ZHU Jin-Yan-1, YANG Jie-1, SHAO Min-2, ZHONG Wei-Gong-1

Scientia Agricultura Sinica. 2014, 47(13): 2485-2493. doi:10.3864/j.issn.0578-1752.2014.13.001

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【Objective】The objective of this study is to analyze the role of cytochrome P450 gene Oscyp71Z2 in resistance to blast in rice, and expose the resistance mechanism mediated by Oscyp71Z2.【Method】Based on the transcriptome profile of the transgenic rice line NJH12 expressing hrf1 from Xoo, a resistance-related gene showing 114.6-fold increase of expression level was identified. The gene is the cytochrome P450 gene, named as Oscyp71Z2 (NCBI No.: Os07g0217600). Oscyp71Z2 coding sequence was cloned from rice cultivar Nipponbare transcript by PCR. To better understand the function of P450 gene Oscyp71Z2, binal transformation vectors pVec8::Oscyp71Z2 for Oscyp71Z2 overexpression were made, pVec8::Oscyp71Z2 plasmid was transformed into Agrobacterium tumefaciens strain EHA105 by freezing and thawing, and Oscyp71Z2-overexpressing rice was obtained by A. tumefaciens method. The selective marker hygromycin phosphotransferase gene hph was detected in T4 Oscyp71Z2-overexpressing rice by normal PCR, and the relative expression level of Oscyp71Z2 in transgenic rice was analyzed by qRT-PCR. Resistance phenotypes to Magnaporthe oryzae in positive Oscyp71Z2-overexpressing rice (T4, T5 and T6) were identified at booting stage. And phytoalexins accumulation of Oscyp71Z2-overexpressing rice (T4) was quantified.【Result】RT-PCR and qRT-PCR data showed that the expression level of cytochrome P450 gene Oscyp71Z2 in hrf1 transgenic rice with resistance to blast was higher than that in wild type rice, which was consistent with the Oscyp71Z2 data from transcriptome profile. Oscyp71Z2 gene cDNA is 1 554 bp, contains two cytochrome P450 domains by bioinformation analysis, which belongs to the CYP71Z subfamily. The full-length cDNA of Oscyp71Z2 was successfully ligated into binary vector pVec8, and transformed into rice Nipponbare genome mediated by A. tumefaciens. The rice growth periods, plant height and other traits in Oscyp71Z2-overexpressing rice were consistent with that in wild type rice. The transcripts of Oscyp71Z2 in Oscyp71Z2-overexpressing rice obviously increased, compared with that in wild type. The resistance degree to panicle blast in wild type was 3-4, showed susceptibility. However, the resistance degree to panicle blast in Oscyp71Z2-overexpressing rice was 1-2, showed resistance or middle resistance. The overexpression of Oscyp71Z2 in rice significantly enhanced the resistance to panicle blast. The accumulation levels of phytoalexin MA and MB in fresh leaves of Oscyp71Z2-overexpressing rice were 328 and 124 ng•g-1, respectively, which were higher than that of 85 and 42 ng•g-1 in wild type fresh leaves, respectively. Furthermore, the relative expression level of four key genes (OsCPS2, OsCPS4, OsKSL7 and OsKSL4) involve in phytoalexin MA and PB synthesis pathway in Oscyp71Z2-overexpressing rice was obviously increased.【Conclusion】The overexpression of cytochrome P450 gene Oscyp71Z2 in rice conferred resistance to panicle blast via activation of phytoalexins biosynthesis pathway.

Screening and Identification of Proteins Interacting with TaMAPK2 in Wheat

YU Tai-Fei-1, 2 , XU Zhao-Shi-2, LI Pan-Song-2, CHEN Ming-2, LI Lian-Cheng-2, ZHANG Jun-Hua-1, MA You-Zhi-2

Scientia Agricultura Sinica. 2014, 47(13): 2494-2503. doi:10.3864/j.issn.0578-1752.2014.13.002

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【Objective】Mitogen-activated protein kinases (MAPKs) play an important role in stress signal transduction process in plant. To provide data for exploring the functional mechanism of TaMAPK2, a wheat cDNA library was constructed and its interacting proteins were screened by yeast two-hybrid system.【Method】The wheat cDNA was used as the template for amplifying the TaMAPK2 gene, and the bait plasmid pGBKT7-TaMAPK2 was constructed. The mixture of recombinant plasmid pGBKT7- TaMAPK2, pGADT7 with wheat cDNA library was introduced into yeast cell AH109. Transformed cells were incubated on SD/-Trp/ -Leu/-His/-Ade plate at 30℃ for 3-5 days before selection of clones and further incubated on SD/Raf/Gal/x-gal for screening blue clones. The candidate proteins that interacted with TaMAPK2 were obtained by sequencing and analyzing the blue clones. Enzyme digestion and ligation method was used to construct the expression vectors of pGADT7-HSP90, pSPYNE-TaMAPK2 and pSPYCE-HSP90. The recombinant plasmids pGADT7-HSP90 and pGBKT7-TaMAPK2 were transformed into yeast competent cells AH109, and the yeast two-hybrid system was used to analyze the interaction between TaMAPK2 and HSP90. The expression vectors pSPYNE-TaMAPK2 and pSPYCE-HSP90 were transformed into wheat protoplasts by using PEG-Ca2+ method. At last the interactions of TaMAPK2 and HSP90 were observed using confocal laser scanning microscope. A real-time PCR testing system was constructed to quantify HSP90 in wheat by the specific primer, designed on the conservative sequences of HSP90 complete genome, and a testing mode based on SYBR Green I technology．The expression patterns of HSP90 gene in response to different abiotic stresses and in different organs were analyzed by quantitative real-time PCR.【Result】 The proteins that interacted with TaMAPK2 were obtained, and most of the candidate proteins involved in signal transduction and immune process. This result suggests that TaMAPK2 possibly plays significant roles in stress signal transduction, transcription regulation of downstream genes and translation process in stress environments. Yeast two-hybrid and BiFc showed that HSP90 protein interacted with TaMAPK2. Subcellular localization analysis revealed that HSP90 is located on the plasma membrane, the cytoplasm and nucleus. The QRT-PCR showed that HSP90 expressed in roots, stems and leaves. The transcripts of HSP90 were more abundant in roots. Real-time PCR showed that the expression of HSP90 was up-regulated by the imposition of high-temperature, low-temperature, high-salt and ABA stresses.【Conclusion】The HSP90 is a widespread family of molecular chaperones. HSP90 can contribute to maintain cellular integrity by folding damaged proteins and maintaining protein conformation. Abiotic stress conditions cause the accumulation of aggregates containing damaged and abnormal proteins in plant. HSP90 is required for the degradation of damaged and misfolded proteins, and mediates expression of transcription factors. This result suggests that TaMAPK2 transmits signals by interacting with other proteins in plant abiotic stress responses.

Review and Inspiration of Plant Proteins Involved in the Transformation Processing of T-DNA Initiated by Agrobacterium

ZHAO Pei, WANG Ke, ZHANG Wei, DU Li-Pu, YE Xing-Guo

Scientia Agricultura Sinica. 2014, 47(13): 2504-2518. doi:10.3864/j.issn.0578-1752.2014.13.003

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Agrobacterium tumefaciens is a kind of Gram-negative soil bacterium, which harbors tumor inducing (Ti) or root inducing (Ri) plasmid. The T-DNA on the plasmid can enter plant cells and integrate into the host genome, and further endows plants new characteristics by stable inheritance. Thus, Agrobacterium tumefaciens, as a most efficient transformation vehicle, has been widely used in genetic engineering study for most dicotyledonous and part of monocotyledonous plants. Agrobacterium-mediated transformation technology has several advantages including simple process, low cost, less gene silence, and few copies of target genes. However, the transferring of target genes into plant genome by Agrobacterium is a complicated biological process, during which a lot of Agrobacterium proteins and plant proteins interact with each other for the mission of importation, transportation and integration of T-DNA. Associated host proteins play important roles almost in each step of the transformation of exogenous genes. For instance, AGPs, rhicadhesin binding protein, and vitronectin-like protein are involved in the Agrobacterium attachment to the surface of plant cells; GTPase and BTI proteins help T-DNA and Vir proteins enter plant cell; Actin, GIP, and VIP assist T-DNA transportation in plant protoplasm; VIP1, KAPa, PP2C, and Roc participate in the targeting and importing of T-DNA complex to plant cell nuclear, histones; VIP1 and VIP2 perform functions on the integration of T-DNA into plant genome. Due to the huge differences between plants in genomics, physiology and biochemistry, overexpression of some plant genes mentioned above do improve the transformation frequency in some plants mediated by Agrobacterium, while that doesn’t work in other plants. It was indicated that VIP1, VIP2, AGP and H2A are closely associated with the Agrobacterium-mediated transformation efficiency of some model plants like Arabidopsis and rice. However, the functions of these proteins in some recalcitrant plants to Agrobacterium infection such as wheat and maize are still undetermined. Thus, some novel proteins or genes associated with T-DNA transformation need to be isolated and identified in different plants. Presently, there are two marked features in the Agrobacterium-mediated transformation of plants. One is that the transformation technology of Agrobacterium-mediated for model plant species such as tobacco, Arabidopsis and rice is almost reaching a perfect level, and another one is that the technique is still difficult to be applied in some economically important crops including wheat, maize and soybean. Therefore, it is necessary to further investigate the roles of plant proteins associated with the transformation network in more plants. In this article, the plant proteins related to Agrobacterium infection and T-DNA delivery reported to date are reviewed to provide some useful information for the improvement of Agrobacerium-mediated transformation efficiency for some recalcitrant plants to be transformed.

Dynamic Changes of Phytohormones as Influenced by Different Plant Growth Substances in a Dwarf-Multi-Tiller Rice Mutant

LIU Qing-1, TONG Jian-Hua-1, SHI Qi-1, PENG Ke-Qin-1, WANG Ruo-Zhong-1, LIN Wan-Huang-1, MohammedHumayunKabir1 , SHEN Ge-Zhi-2, XIAO Lang-Tao-1

Scientia Agricultura Sinica. 2014, 47(13): 2519-2528. doi:10.3864/j.issn.0578-1752.2014.13.004

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【Objective】 The objective of study is to analyze the contents of different phytohormones at different developmental stages of a dwarf-multi-tiller rice mutant and its wild-type, and also compare the changes of morphological traits when treated by different plant growth substances. 【Method】 A T-DNA inserted dwarf-multi-tiller rice mutant (designed CA648) and its wild-type Zhonghua 11 (designed ZH11) were used as materials. HPLC was employed to analyze GA1, GA4, IAA, Z and ABA levels at different developmental stages (3rd leaf, 5th leaf, mid-tillering and booting stage), and compare the phytohormones content between CA648 and ZH11 in different organs (root, culm and leaf). In addition, endogenous IAA, Z and ZR were quantified at the booting stage in the different organs (root, stem, leaf and basal node) by using HPLC-MS/MS method, and the ratio of IAA to Z+ZR was used to compare between CA648 and ZH11. The changes of agronomic traits such as plant height and tiller numbers after the plants treated by spraying different plant growth substances (GA3, IAA, Z and paclobutrazol) were also examined.【Result】The trend of phytohormonal change between CA648 and ZH11 varied in different organs at different developmental stages. The content of GA1+4 in the culm and root of CA648 was higher than in ZH11, while there was no obvious difference in leaf at 3rd leaf stage, and the trend of GA1+4 content in culm (or stem) and root were raised but in leaf it was descended in ZH11 from 5th leaf to heading stage. However, a contrary trend was observed in CA648 for GA1+4 content in culm, root and leaf from 5th leaf to heading stage. IAA content showed no significant difference at 3rd leaf stage between CA648 and ZH11. A higher level of IAA in culm but a lower level in leaf sample of CA648 than ZH11 at the 5th leaf and mid-tillering stage were observed. At booting stage, the same part of root, stem or leaf had almost the same level of IAA, no matter the samples were from CA648 or ZH11. The level of Z was lower in CA648 than ZH11 in root or stem at 3rd leaf stage. At booting stage, CA648 had higher Z+ZR content in basal node or stem, but it had a lower level Z+ZR in leaf than that in ZH11. The ratio of IAA to Z+ZR in sample of leaf or stem from CA648 was greater than that of ZH11, but in sample of basal node or root had the contrary trend. ABA content in CA648 was higher at 3rd leaf and 5th leaf stages, but lower at booting stage than in ZH11. Results of spraying plant growth regulators showed that the plant height of CA648 is sensitive to exogenous GA3, IAA, Z or paclobutrazol but the multi-tiller characteristic were stable. In comparison with control (sprayed H2O), all treatments by application of exogenous GA3, IAA or Z could increase the plant height of CA648, and GA3 exhibited the highest sensitivity, followed by IAA and Z. But the plant height of CA648 and ZH11was decreased in case of paclobutrazol application. Although both CA648 and ZH11 plant height could be regulated by exogenous GA3 or paclobutrazol application, CA648 showed hypersensitive reactions to exogenous GA3. The plant height of CA648 could be rescued back to or even higher than ZH11 when 288 µmol•L-1 or higher level of GA3 was sprayed. It was different to plant height, the multi-tiller trait of CA648 was insensitive to exogenous plant growth substances, and it always produced more tillers than ZH11, being irrelevant to plant growth substance treatment. 【Conclusion】 The contents of GA1, GA4, IAA, Z, ZR and ratio of IAA to Z+ZR in CA648 are more beneficial than that of ZH11 to produce a dwarf-multi-tiller trait, therefore, the level of phytohormones contents and their ratio can reflect the physiological characteristics of CA648. Plant growth substances application caused different effects on plant height and tiller number. Spraying GA3, IAA, Z or paclobutrazol can change CA648’s height, while the multi-tiller trait is unaffected.

Differential Response of Floret Opening to Exo-Methyl Jasmonate Between Subsp.Indica and Subsp. Japonica in Rice

YAN Zhi-Qiang-1, XU Hai-1, MA Zuo-Bin-1, 2 , GAO Dong-Chang-1, XU Zheng-Jin-1

Scientia Agricultura Sinica. 2014, 47(13): 2529-2540. doi:10.3864/j.issn.0578-1752.2014.13.005

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【Objective】 hybrids between indica and japonica rice can produce significant hybrid vigor or heterosis, application of which can improve yields and tolerance to the biotic and abiotic stresses of rice. One of the major factors limiting the application of heterosis in rice is the big difference of flowering time in a day between the two subspecies. Indica rice often blooms early, but japonica rice blooms later. The flowering asynchronism will reduce the yields of hybrid seeds, enhance the cost of hybrid seed production, and further constrain the application of heterosis between the two subspecies. Previous results show that spraying methyl jasmonate (MeJA) can effectively promote rice flowering in the day. But the sensitivity of flowering time of rice varieties to MeJA remains obscure. In this study, the differential responses of the floret opening to MeJA between indica and japonica rice were investigated, which will provide a theoretical basis for improving the yields of the inter-subspecies hybrid rice seed production and enhancing the possibility to utilize the heterosis between indica and japonica rice directly.【Method】 To investigate the dynamics of floret opening, six varieties of subsp. indica and japonica were respectively, planted in the experimental station of Rice Research Institute at Shenyang Agricultural University in 2012 and 2013. Three concentrations of MeJA, 0.04 mmol•L-1, 0.4 mmol•L-1, and 4 mmol•L-1, were sprayed on the panicle and top leaves of the rice plants at 17:00 last afternoon, 6:00 and 8:00 in the morning, respectively. Water, no addition of MeJA, was used as control (CK). The flowering-begin-time (FBT), the vigorous-flowering-time (VFT), the flowering-end-time (FET), the time from the beginning of flowering to vigorous flowering (BVT), the time from the beginning of vigorous flowering to flowering end (VET), the time from the beginning of flowering to end (BET) of indica and japonica rice were investigated after application of MeJAs at different times. Criteria for determining the flowering time were as follows: The time of the first flower opening, the time of the most flowers opening, and the time of all the flowers closed were used as the measurement of the FBT, the VFT, and the FET, respectively. The experiment was designed on the randomized block with three replications.【Result】 The concentration of MeJA could significantly promote the flowering time of indica rice compared to the no MeJA treatment (CK). There was no significant difference among the MeJA treatments. Flowering time in a day of japonica rice only responded to the high (4 mmol•L-1) and medium (0.4 mmol•L-1) concentrations of MeJA. There was a significant difference between high concentration and medium concentration of MeJA treatments. The concentration of MeJA had no significant effect on the BVT and VET of both indica rice and japonica rice in contrast to CK. However, the application of MeJA, no matter of the concentration, could obviously extend the BET in indica rice. The spraying time did not affect the flowering time of indica rice. In contrast to indica rice, spraying MeJA at different times of a day, such as 5:00 pm last afternoon, 6:00 am, and 8:00 am in the morning, had significant effects on the flowering time of japonica rice. The results also indicated that high concentration of MeJA could promote the flowering time of indica and japonica rice equally. But lower (0.04 mmol•L-1) and medium (0.4 mmol•L-1) concentrations of MeJA were more effective on accelerating flowering of japonica rice than that of indica rice.【Conclusion】The initial time of floret opening in all indica and japonica rice varieties tested was significantly promoted by applying 4 mmol•L-1 and 0.4 mmol•L-1 of MeJA. The flowering time in a day of six indica varieties, not all, was response to the low dosage of MeJA (0.04 mmol•L-1), suggesting that indica rice was more sensitive to the MeJA treatments compared to japonica rice. The varieties of indica rice responded to MeJA more quickly than japonica rice. Longer time was needed for varieties of japonica rice to flowering as the results of applying MeJA. Treatment with high concentration (4 mmol•L-1) of MeJA at 17:00 last afternoon was the best combination to promote flowering of japonica rice. The optimal combination to promote flowering of indica rice varied from cultivar to cultivar. The most economic and efficient combination was 0.04 mmol•L-1 MeJA sprayed in the morning.

Study on Climatic Suitability for Winter Rapeseed Planting in Arid and Cold Regions in North China

ZHOU Dong-Mei-1, ZHANG Ren-Zhi-1, 2 , SUN Wan-Cang-2, 3 , ZHANG Jun-1, 2 , WANG He-Ling-4

Scientia Agricultura Sinica. 2014, 47(13): 2541-2551. doi:10.3864/j.issn.0578-1752.2014.13.006

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【Objective】By analyzing the relationship between rapeseed cultivation and climatic conditions of arid and cold regions in North China, dividing regions with climatic adaption for winter rapeseed, this paper aimed to provide a reference for improving winter rapeseed production layout and restructuring agricultural construction. 【Method】 Based on experimental data of rapeseed varieties in different zones and climate data of long sequences, using GIS spatial analysis functions and maximum entropy models, the relational potential climatic factors for the distribution of winter rape growing were screened, meteorological factors were used to establish the potential of spatial database based on the DEM method of small grid reckoning. This study analyzed the relationship between rapeseed cultivation and climatic conditions in arid and cold regions, simulated the potential distribution probability and divided grow regions with climatic adaption for winter rapeseed.【Result】The total contribution rate of the potential to meteorological factors to the distribution of winter rape cultivation was 0.89, and according to the size of the contribution rate, there were 5 major climatic factors affecting the growth distribution of winter rapeseed, including annual average temperature, negative accumulated temperature, extreme low temperature, minimum temperature of the coldest month and the average temperature of the coldest month. The potential distribution probability of winter rapeseed ranges was 0-0.84 in arid and cold regions. Growth regions were divided into four levels according to climatic adaption: unsuitable, less suitable, suitable and optimum suitable. The northern boundary of winter rapeseed cultivation have reached the southern Jilin, Inner Mongolia, and southern Xinjiang as new boundaries, the northern boundary has expanded northward about 1 200 km, from the 39°N raised to 45°N compared with the traditional winter rapeseed planting northern boundary. 【Conclusion】 This study revealed the main climate factors affecting growth distribution of winter rapeseed, divided suitable growth areas for winter rapeseed of arid and cold regions in regional scale. About more than 50% of region can be planted with winter rapeseed in northern and cold regions of China. It was proved that it is possible to expand winter rapeseed northwards into arid and cold regions in North China. Winter rapeseed will be an important oil crop in northern and cold regions of China, and it will break the existing traditional winter rapeseed planting division, changing the layout of winter rapeseed production structure.

Molecular Cloning of T-DNA Targeted Genes of Ustilaginoidea virens Pathogenicity-Defective Mutant Strain B-1015

HUANG Lei-1, 2 , HU Jian-Kun-2, YU Mi-Na-2, YU Jun-Jie-2, WANG Ya-Hui-2, ZHENG Meng-Ting-2, ZHENG Rui-2, LIU Yong-Feng-1, 2

Scientia Agricultura Sinica. 2014, 47(13): 2552-2562. doi:10.3864/j.issn.0578-1752.2014.13.007

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【Objective】 The objective of this study is to analyze the biological phenotypes of the pathogenicity-defective mutant strain B-1015, to clone its T-DNA integration flanking sequences, and finally to reveal the functions of targeted genes in pathogenesis of Ustilaginoidea virens. The study will contribute to better understanding of the molecular pathogenesis of U. virens. 【Method】 Biological phenotypes of B-1015 were analyzed by testing growth rate, sporulation ability, spore germination rate and pathogenicity. The copy numbers of T-DNA inserted into B-1015 were identified by Southern blot. The flanking sequences of T-DNA were cloned by hiTail-PCR and the whole gene sequences of the T-DNA targeted genes were cloned by RACE PCR. The genes expression were detected by RT-PCR. 【Result】By comparing with wild type strain P1, the sporulation ability and growth rate of the B-1015 were significantly declined, but the spore germination rate had no significant difference. The inserted mutant lost its pathogenicity in rice. Molecular detection analysis confirmed that the B-1015 was a stable single T-DNA insertional mutant. The T-DNA flanking sequences were obtained by hiTail-PCR. Two genes, Uvt-1015R and Uvt-1015L, were cloned by RACE PCR. Gene sequence analysis showed that Uvt-1015R contained a 948 bp open reading frame, which could encode a 295 amino acid protein, a 341 bp 5′-UTR and a 271 bp 3′-UTR. Another gene, Uvt-1015L, comprised a 351 bp open reading frame coding a 116 amino acid protein, a 31 bp 5′-UTR and a 174 bp 3′-UTR. By searching in the known protein, the Uvt-1015R and Uvt-1015L genes showed no homology of any protein. Sequence alignment showed that the flanking sequences of T-DNA were non-adjacent in the wild type genome and the T-DNA disrupted the two genes sequence. RT-PCR analysis confirmed that compared to P1 strain, the transcripts of Uvt-1015R was significantly decreased, and the transcripts of Uvt-1015L was not detected any more. 【Conclusion】 In B-1015, both the T-DNA insertion and chromosomal rearrangement caused important biological phenotype mutant, thus leading to the loss of pathogenicity.

Molecular Characterization of the Negative Regulator PXO_02944 in Virulence, Extracellular Polysaccharide Production and Biofilm Formation in Xanthomonas oryzae pv. oryzae

LI Xiao-Tong, YANG Feng-Huan, LIANG Shi-Min, TIAN Fang, CHEN Hua-Min, HE Chen-Yang

Scientia Agricultura Sinica. 2014, 47(13): 2563-2570. doi:10.3864/j.issn.0578-1752.2014.13.008

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【Objective】Xanthomonas oryzae pv. oryzae (Xoo) is the causing pathogen of bacterial blight of rice. Xoo can use extracellular polysaccharide (EPS) and enzymes, T3SS and its effectors for sucessful infection of rice. It has been shown that cyclic di-GMP signaling plays an important role in regulation of virulence in Xoo. PXO_02944, the GGDEF, EAL and REC domain containing protein, belongs to c-di-GMP signaling protein family. The objective of this study is to demonstrate the structure and function of the PXO_02944 in regulation of virulence expression of Xoo. 【Method】 Based on the sequences of PXO_02944, the specific primers were designed to amplify the full length of gene using the genomic DNA of the wildtype strain PXO99A as the template. In silico analysis of the GGDEF and EAL domains of PXO_02944 with the conserved ones from other bacteria were done. Then the upstream and downstream fragments of PXO_02944 were amplified, and ligated to vector pK18mobsacB to get the plasmid pK2944. Then a Gm resistance gene (GmR) was inserted into pK2944 resulting in plasmid pK2944-GmR to construct the gene deletion mutation by marker exchange. The full length of PXO_02944 was cloned and ligated into the vector pHM1, and the plasmid was electroporated into ΔPXO_02944 to get the complementory strain. The virulence, EPS production, enzymatic activities, biofilm formation and motility of ΔPXO_02944 were tested compared with PXO99A, ΔPXO_02944 and complemented strain. And the expression of EPS production and virulence related genes were analyzed.【Result】Bioinformatic analysis indicated that PXO_02944 was a response regulator of two-component regulatory system (TCS), which contained the REC input domain and the GGDEF and EAL output domains. In the mutant, PXO_02944 was deleted by replacing of the GmR. The mutation in PXO_02944 led to significant increases in bacterial virulence on rice, EPS production, biofilm formation, hrpG and gumG gene transcripts compared to PXO99A. Interestingly, no changes in activities of cellulase and xylanase and flagellar motility were observed in ΔPXO_02944. The deficiency above in the mutant could be restored to the wild-type level by in trans expression of the PXO_02944. 【Conclusion】 PXO_02944 might function as a negative regulator in bacterial virulence, EPS production and biofilm formation in Xoo.

Histochemical Assays and Signaling Pathway Analysis of β-Aminobutyric Acid Induced Resistance in Potato Against Phytophthora infestans

WANG Jing, WANG Hai-Xia, TIAN Zhen-Dong

Scientia Agricultura Sinica. 2014, 47(13): 2571-2579. doi:10.3864/j.issn.0578-1752.2014.13.009

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【Objective】 β-aminobutyric acid (BABA) can effectively induce defense responses in potato against late blight disease by means of spray in field or on detached leaves. The objective of this study is to investigate the histochemical aspects of BABA induced resistance (BABA-IR) and signaling pathway involved in BABA-IR in potato. The present study will provide knowledge for further elucidating the mechanisms of BABA-IR.【Method】Potato plants were sprayed with 4 mmol?L-1 BABA or distilled water (Mock, as control). Three days after treatment, the sprayed leaves were detached and inoculated with Phytophthora infestans. Then leaf discs were punched out from attached leaves at different time points after inoculation and used for histochemical staining and hypersensitive response (HR) observation. Diaminobenzidine (DAB) staining method was used to detect hydrogen peroxide (H2O2) generation around inoculated site. Callose deposition and HR were detected by aniline blue staining combined with fluorescence microscopic observation. For investigating the signaling pathway in which BABA induced late blight resistance involved, transgenic potato plants which disturbed the jasmonic acid (JA) or salicylic acid (SA) signaling pathway by interferencing StCOI1 or overexpressing NahG were used. 【Result】 No deposition of H2O2 was observed when Mock and BABA pretreated leaves were inoculated with water. While H2O2 deposition appeared around inoculation sites on both Mock and BABA pretreated leaves 24 h after P. infestans inoculation. It was obvious that BABA pretreated potato leaves accumulate H2O2 strongly and early ahead of Mock (control) 12 h after P. infestans inoculation. No obvious callose deposition was observed when Mock and BABA pretreated leaves were inoculated with water. But strong callose deposition was observed around the P. infestans inoculation sites in BABA pretreated potato leaves compared to that of Mock leaves. Fluorescence microscopic observation showed that HR occurred around the P. infestans inoculation sites on BABA pretreated leaves as early as 24 h. Although HR appeared on both Mock and BABA pretreated leaves, high frequency (85%) of HR occurred around the inoculation sites on BABA pretreated leaves, which was about 55% higher than it occurred on the Mock leaves. Treatment with BABA could not effectively induce late blight resistance in the potato leaves overexpressing NahG, while it was capable of inducing resistance in the StCOI1 silenced potato leaves, which meant that BABA-IR needs SA signaling pathway. 【Conclusion】 Various of defense responses, including H2O2 accumulation, callose deposition and HR, were rapidly and strongly activated in BABA pretreatment potato leaves after P. infestans inoculation. SA signaling pathway involved in BABA-IR in potato against P. infestans.

Study on Wheat Yield Stability in Huaibei Lime Concretion Black Soil Area Based on Long-Term Fertilization Experiment

CHEN Huan-1, CAO Cheng-Fu-1, KONG Ling-Cong-1, ZHANG Cun-Ling-2, LI Wei-1, QIAO Yu-Qiang-1, DU Shi-Zhou-1, ZHAO Zhu-1

Scientia Agricultura Sinica. 2014, 47(13): 2580-2590. doi:10.3864/j.issn.0578-1752.2014.13.010

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【Objective】The research was conducted to explore the responsive mechanism of wheat yield stability to long-term fertilization. 【Method】 Based on long-term fertilization experiment in Yangliu village of Anhui province, the trend of change in wheat average yield, annual fluctuation of wheat yield and soil nutrients content in 5 fertilization patterns were investigated: non-fertilization (CK), application of single chemical fertilizer (NPK), application of single organic fertilizer (M), mixed application of organic and chemical fertilizer with the same amount of nitrogen (MNPK), mixed application of organic and chemical fertilizer with the larger amount of nitrogen (HMNPK).【Result】It was discovered that wheat yield in CK showed a declining trend by 5.81 kg•hm-2•a-1, while fluctuated increasing emerged in fertilization treatments. Wheat yield trend line of HMNPK was in the first place, but MPK had been chasing after by 9.75 kg•hm-2•a-1. Wheat yield trend line of NPK was higher than M in the earlier stage of experiment, whereas it was caught up by M after 22 years. In respect of wheat average yield of 32 years, HMNPK and MNPK were higher than other fertilization treatments, with 5 544.3 kg•hm-2 and 5 200.6 kg•hm-2, respectively; NPK took the third place, by increasing 614.6% of that in the non-fertilization treatment; wheat yield increasing of M was the lowest, however, no obvious difference was found between M and NPK. The contribution of soil capacity in lime concretion black soil had been decreasing in early 10 years then stopped and kept stable at 10%; the contribution ratio of fertilizer had been increasing in early 10 years, and then maintained dynamic balance at the level of 80%-90%. It enhanced the coefficient of variation (CV) and reduced sustainable yield index (SYI) without fertilization, which made yield stability the worst; the wheat yield stability of HMNPK and MNPK was better than NPK which was better than M. Compared with CK, fertilizer application increased the content of total nitrogen, organic matter, available phosphorus and available potassium in soil. Addition of organic fertilizer intensified the content of soil total nitrogen and organic matter. Available phosphorus content was related to application of chemical fertilizer. Available potassium content was at the relatively higher level in M, but the differences with other fertilization treatments were not significant. The contents of total nitrogen, organic matter and available phosphorus were significantly positively correlated with yield in Huaibei lime concretion black soil (P＜0.01).【Conclusion】Fertilizer application increased wheat yield significantly in Huaibei. The yield of HMNPK and MNPK were at the higher level, but the yield gap between the two treatments was shortening with growing years. Wheat yield of NPK was higher than M in earlier stage of experiment, while it was caught up with M after 22 years. Compared with CK, mixed application of organic and chemical fertilizer conduced to enhance wheat yield stability and sustainability, NPK took the second place, M placed the last. Fertilization increased soil nutrient content: organic fertilizer enhanced organic matter, total nitrogen, while chemical fertilizer enhanced available phosphorus. Wheat yield correlated positively with total nitrogen, organic matter and available phosphorus significantly. In a word, mixed application of organic and chemical fertilizer is the best fertilization mode in Huaibei lime concretion black soil area, which made soil nutrient more balanced, wheat yield more stable and agricultural ecosystem quality better.

Characterizing Variation of Topsoil Particle Size Distribution Based on Fractal Theory and Geostatistics

ZHANG Shi-Wen-1, 2 , ZHANG Li-Ping-2, YUAN Jun-3, SHEN Zhong-Yang-2, CHEN Xiao-Yang-1, YE Hui-Chun-2, HUANG Yuan-Fang-2

Scientia Agricultura Sinica. 2014, 47(13): 2591-2601. doi:10.3864/j.issn.0578-1752.2014.13.011

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【Objective】 This study aimed to explore the methodology which can help fully reveal the characteristics of soil particle size distribution (PSD) on multi-angle and multi-scale, and to find more simple and comprehensive means of quantitative analysis and evaluation of soil quality and its evolution. 【Method】 The study calculated the volume fractal dimension (Dv) and analyzed the variation characteristics of soil particle size distribution based on fractal theory, traditional statistics, separated soil particles Dv log-log graph and geostatistics from point and regional scale. 【Result】 Cumulative volume percentage of soil particles with diameter＜10 μm showed a significant positive correlation with Dv while that of soil particles with diameter＞50 μm was negatively related to Dv. The smaller soil particles Dv was, the coarser soil texture was. The end portions of log-log scatter plot for the maximum and minimum Dv bent downward with the fitting straight lines close to major changes, the value of R2 are above 0.9, and the fitting results are ideal. The separation of Dv contained the entire soil particle size distribution (PSD) of the degree of change. There were some differences of soil particles Dv between different groups of soil organic matter content, and soil particles Dv was able to objectively characterize the changes of soil quality of a farmland. With elevation increasing, the performance of soil particles Dv was relatively complex. The mean value of soil particles Dv for cinnamon soil was maximum, mean was minimum for fluvo-aquic soil. The results of variance analysis showed that the difference in Dv values between grain field, garden land and vegetable land was not obvious. Prediction results with regression kriging were more accurate based on the analysis results about the relationship between soil particle Dv and environmental variables. Spatial distribution pattern of the regional and sampling Dv was consistent, and objectively reflected the variation characteristics of PSD. 【Conclusion】 Methodology established in the study can reflect the characteristics of PSD from multi-angle and multi-scale. The study results matched the actuality, and soil particles Dv can be used as a means to characterize and evaluate the soil quality and its changing process.

Bioinformatics and Expression Analysis of the LysM Gene Family in Apple

ZHOU Zhe, ZHANG Cai-Xia, ZHANG Li-Yi, WANG Qiang, LI Wu-Xing, TIAN Yi, CONG Pei-Hua

Scientia Agricultura Sinica. 2014, 47(13): 2602-2612. doi:10.3864/j.issn.0578-1752.2014.13.012

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【Objective】 The objective of this study is to lay a basis for the further functional identification and application of LysM genes by bioinformatics and expression analysis. 【Method】 Based on apple genome database GDR and FEM-IASMA, the sequences of LysM family genes were identified and numbered in apple. Basic information of the MdLysM amino acid sequence was predicted by ExPASy Proteomics Server, the prediction of the sublocation was finished by the WoLF PSORT. A phylogenetic tree was constructed using the MEGA5 software. The plaza was used to draw the structure of the genes. Information about the chromosome location was obtained from the GMDO. Finally, the map of the chromosome location of the 39 MdLysM genes was accomplished through MapInspector. In addition, real-time qRT-PCR was used to determine the expression pattern of these genes, and the significance of difference was analyzed by SPSS. 【Result】A sum of 39 LysM genes were identified systematically in apple. The number of amino acid of the 39 MdLysM proteins ranges from 241 to 1 119. The isoelectric point distributed from 4.70 to 9.60. Sublocation results indicated that the LysM proteins in apple exist in the nucleus, cytoplasm, chloroplast, vacuole and extracellular matrix. They were classified into three groups (group A, B and C), of which group A was divided into three subgroups according to the phylogeny relationship and gene structures, this implies that function differentiation may have happened. The predicted results of the protein domains were congruent with the clustering in phylogenetic tree. The results of chromosomes location indicated that among apple’s all 17 chromosomes, 13 chromosomes have LysM genes located on and the distribution of these genes was not homogeneous. Compared to other chromosomes, chromosome No. 4 contains more genes with a number of 9, while none of the identified genes localized on chromosomes No.1, No.5, No.7 and No.8. Ten pairs and 1 group of paralogs were identified in apple LysM gene family. Tandem and segmental duplication exist among them, and they are the main power for the expansion of apple LysM gene family. The expression patterns of the 39 MdLysM genes in roots, stems, leaves, flowers and fruits were determined by quantitative real-time PCR, and the result shows that their expression can be detected in all of the five organs. Their expression patterns are diverse which indicate that they may play different roles in different organs.【Conclusion】Thirty-nine LysM gene family in apple were identified in genome-wide scan. These genes were classified into three groups and distributed on 13 chromosomes with different tissues expression patterns. These results were significant to forecast the functions of LysM gene family in apple.

Isolation of Florigen Gene PdFT and Its Effects on Flowering of Tree Peony（Paeonia delavayi Franch.）

ZHU Fu-Yong, LIU Chuan-Jiao, XUE Jing-Qi, WANG Shun-Li, ZHANG Ping, REN Xiu-Xia, ZHANG Xiu-Xin

Scientia Agricultura Sinica. 2014, 47(13): 2613-2624. doi:10.3864/j.issn.0578-1752.2014.13.013

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【Objective】The objective of this study was to clone the homolog of FT gene from tree peony and analyze the expression pattern and the potential function of flowering locus T (FT) of flowering in Paeonia delavayi Franch..【Method】The homologous FT gene, PdFT, was cloned by reverse transcript polymerase chain reaction (RT-PCR). The expression patterns of PdFT in different organs, developmental stages of flowering and treatments of P. delavayi Franch. were analyzed by using qRT-PCR and the relationship between the PdFT expression level and the different states of flower bud development was also studied. At the same time, PdFT was inserted into vector pET-28a and expressed in E. coli BL21 (DE3). 【Result】The full length of open reading frame (ORF) of PdFT was 522 bp, encoding 173 amino acid residues. The GenBank accession number is KF113360. Alignment of the AA sequences revealed that PdFT contains a conserved domain, which possesses the characteristics of the PEBP family. The result also suggested that PdFT shares high identity with FT proteins from other plants. qRT-PCR analysis of PdFT expression pattern in different organs showed that the highest expression levels were appeared in the buds of P. delavayi, followed by roots, stems and leaves. The expression pattern of PdFT from bud swelling to big-like flower-bud showed a decreased trend. The expression level of PdFT under long day condition was higher than that under short day condition. Cool treatment could decrease PdFT expression in roots and stems. The expression of PdFT under different floral conditions showed difference. The expression of PdFT in abortive flower buds was lower than that in normal bud. GA3 treatment and removing leaves could increase the expression of PdFT. The results of SDS-PAGE showed that the expressed protein in E. coli BL21 (DE3) was consistent with the size of expected. 【Conclusion】The PdFT isolated from P. delavayi Franch. is highly homologous with the other FT genes isolated from other species and belongs to FT subfamily. The higher expression of PdFT in buds suggested that it may play a role in regulating flowering in tree peony. PdFT gene cloning and expression analysis will lay a genetic basis for investigating the molecular mechanism of flowering in P. delavayi Franch.

Extraction Conditions Optimization of Main Organic Acids from Fruits

PANG Rong-Li, FANG Jin-Bao, GUO Lin-Lin, XIE Han-Zhong

Scientia Agricultura Sinica. 2014, 47(13): 2625-2633. doi:10.3864/j.issn.0578-1752.2014.13.014

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【Objective】This study aimed at investigating an effective extraction method of main organic acids(citric acid, tartaric acid, malic acid, succinic acid) from fruits.【Method】Using citrus, grapes and other fruits as test materials, the aqueous ethanol solution as the extraction agent, and using ultrasonic extraction method, the effect of extraction on the extraction agent (ethanol concentration, ratio of water extraction and organic acid stability, different concentrations of ethanol extraction, effect of 10% ethanol soluble extractant and the main organic acid stability and so on), extraction conditions (extraction method, ultrasonic extraction time and so on) were studied for optimization. Technical parameters for optimized extraction conditions (the detection limit, precision, repeatability, accuracy) were tested. The measuring results by using this method were compared with analytical results with the existing national standard method.【Result】The suitable method for organic acid extraction in fruits is the ethanol ultrasonic extraction method: Samples were extracted for 30 min with 10% ethanol solution in ultrasonic extractor. Then sulfuric acid solution was added, the concentration of sulfuric acid contained in solution was the same as in flow consistent solution. Finally constant volume was made with ethanol solution, then mixed and filtered. The filtrate was filtered by 0.22 μm aqueous membrane needle filter for ion chromatography analysis. All the main organic acids could be extracted completely. The main organic acids didn’t change to lactic acid and acetic acid at least in 15 days when the extract was placed under natural conditions. The optimized extraction method is high precision (RSD 0.28%-1.20%), repeatability (variation coefficient of 0.236%-3.02% (n=5)) and high accuracy (recovery 88.9%-101.2%). The method of organic acids in the determination results was significantly higher than the existing national standard results.【Conclusion】The method was rapid, simple, accurate, reproducible, and suitable for the determination of the contents of main organic acid in fruits.

Effect of Pre-Emulsified Emulsion Treated with Ultrasound on Qualities of Low-Fat Frankfurter-Style Sausages

ZHAO Ying-Ying, ZOU Yu-Feng, WANG Peng, CHEN Lin, LI Ke, XU Xing-Lian

Scientia Agricultura Sinica. 2014, 47(13): 2634-2642. doi:10.3864/j.issn.0578-1752.2014.13.015

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【Objective】Pre-emulsified sodium caseinate-soybean oil emulsion prepared with pulsed ultrasound was used to replace pork back fat in frankfurter-style sausages.【Method】Effect of ultrasound pre-emulsified emulsion with different substitution rates (25%, 50%, 75%, and 100%) on the chemical composition, color parameters (L*, a*, b*), textural properties (hardness, chewiness, springiness, cohesiveness, resilience), water- and fat-binding (WFB) capacity (cooking loss, expressing loss), water distribution and microstructure of frankfurters was performed in this study. 【Result】With the increasing of substitution rate, the moisture of frankfurters became larger, fat content and energy value became smaller (P＜0.05). There was no significant difference among frankfurters in ash and protein content (P＞0.05). L* value increased and a* value decreased significantly (P＜0.05) with the increasing of substitution rate. Both of hardness and chewiness decreased as substitution rate increased but significantly higher than the control (P＜0.05). Springiness, cohesiveness and resilience significantly increased with the increasing of substitution rate (P＜0.05). Sausages formulated with pre-emulsions (75%, 100% replacer) had similar (P＞0.05) springiness but higher resilience with the control (P＜0.05). Compared with the control, sausages with the substitution rate of 50%, 75% and 100% had higher cohesiveness (P＜0.05). Cooking loss and expressing loss significantly increased with the increasing of substitution rate but lower than the control (P＜0.05). T23 relaxation time increased from 57.22 ms to 64.57 ms, immobile water increased and bulk water decreased. Frankfurters formulated with ultrasound emulsion had homogeneous fine three-dimensional network textural property with smaller emulsion droplet. 【Conclusion】Ultrasound treatment can produce much smaller pre-emulsified emulsion droplets than conventional homogenization. Good water and fat absorption and binding capacities and eating qualities can be achieved by incorporating ultrasound pre-emulsified emulsion stabilized using sodium caseinate in frankfurter-style sausages.

Effect of Seed Ultra-Drying Storage on Growth and Resistance of the Medicago sativa Seedlings Affected by Alkaline Salt Stress

HUO Ping-Hui, LI Jian-Feng, SHI Shang-Li, ZHANG Shu-Qing

Scientia Agricultura Sinica. 2014, 47(13): 2643-2651. doi:10.3864/j.issn.0578-1752.2014.13.016

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【Objective】 The objective of the experiment is to study the effect of ultra-drying storage on emergence, growth and resistance of alfalfa (Medicago sativa L. cv. Longdong) seedlings derived from seeds with different moisture contents that grew under alkaline salt stress. 【Method】 Alfalfa seeds were packaged into porous nylon bags, placed into hermetic desiccator and buried in cooled silica gel that diurnally dried at 120℃ and temporally (0, 12, 24, 48, 72, 96, 120, 144 and 216 h) differently ultra-dried, then the seed moisture contents were reduced to 9.03% (CK), 7.09%, 6.93%, 6.36%, 5.72%, 5.46%, 5.18%, 4.97% and 4.59%, respectively. The ratio between seeds and silica gel was 1﹕10 (w/w). All the treated seeds were sealed in aluminum foil packages and stored in a desiccator fulfilled with dried silica gel at ambient temperature. After 1-year storage, NaHCO3 and Na2CO3 were mixed at the mole ratio of 9﹕1 to imitate the typical stress conditions, and 15 mmol•L-1 alkaline salt stresses was used to conduct the pot culture with sand using Hoagland solution. 【Result】 The results showed that no significant acceleration of seed ultra-drying storage was observed in seedling emergence, shoot height, root elongation and nodule formation. On day 30, no significant seedling emergence difference was found between 7.09%, 6.93%, 6.36%, 5.72% and 5.46% treatments and the control, while as the decrease of seed moisture contents, seedling emergence in 5.18%-4.59% treatments was found significantly lower than that of the control. On day 60, no significant difference was found in shoot height among all the treatments and the control, together with the nodule number of all treatments except the significantly low level of 4.59% treatment which was only 21.41% of the control. On day 60, root length of 7.09% and 4.97% treatments was significantly higher than the control while no significant difference was found between other seed moisture contents and the control. Moderate seed ultra-drying treatment promoted plant biomass, root volume and individual leaf number. Fresh weight of above ground and under ground parts presented a similar tendency: the value of 6.93%, 6.36%, 5.72% and 4.97% treatments was significantly higher, which were 125.08%-147.84% and 128.36%-271.11% times of the control, respectively. As for the dry weight, only 6.93% treatment was found significantly higher, which were 169.75% and 370.16% times of the control, for above ground and under ground parts, respectively, and all the other treatments were found insignificant compared with the control, except the significant low level of 4.59% treatment in above ground dry weight. On day 60, root volume of 6.93% and 6.36% treatments were 175.68% and 189.21% times of the control, and individual leaf numbers of 6.93%, 6.36% and 4.97% treatments were found significantly higher than the control. Plant root activity increased to 137.45%-199.62% in 6.93%, 6.36%, 5.72%, 5.46%, 5.18% and 4.97% treatments, plant soluble sugar content increased to 176.76%-294.20% in 5.72%, 5.46%, 5.18% and 4.97% treatments, respectively, compared with control. Chlorophyll content of all ultra-dried treatments was 137.82%-211.76% times of the control, while MDA content of all ultra-dried treatments was only 4.66 %-51.69% of the control.【Conclusion】 Moderate seed ultra-drying and storage accelerated plant biomass, root volume, leaf growth and the formation of chlorophyll, and increased root activity and seedling resistance under alkaline stress, indicating that as a kind of seed pre-treatment method, moderate seed ultra-drying promoted the seedling emergence and growth under alkaline stress conditions.

Identification and Virulence Assessment for a Strain of Brucella abortus

DING Jia-Bo-1, WANG Fang-1, YANG Hong-Jun-2, WANG Nan-1, ZHU Liang-Quan-1, GU Jin-Hua-1, ZHANG Guang-Chuan-1, WANG Hai-Guang-1, ZHAO Peng-3, CHENG Jun-Sheng-1, MAO Kai-Rong-1, FENG Yu-3

Scientia Agricultura Sinica. 2014, 47(13): 2652-2658. doi:10.3864/j.issn.0578-1752.2014.13.017

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【Objective】The objective of this study is to explore the biological characteristics and assess the virulence of a B. abortus isolate (strain 343).【Method】The culture of B. abortus 343 was diluted and cultivated to single clone on Tryptic Soy Agar (TSA) for morphological observation, and it was then stained with Gram’s and Koch’s methods. Biochemical characteristics tests (including the growth ability test on thionine and alkaline azaleine, the dependence on CO2, and H2S release test) , single phase specific serum (B. abortus specific anti-serum A, B. melitensis specific anti-serum M and Brucella rough-type specific anti-serum R) agglutination tests and AMOS-PCR were used to identify its comprehensive characteristics. Mice and guinea pigs were infected with a certain dose of B. abortus 343 to determine the virulence of the isolate. The survival time of B. abortus in mice, the isolated number per gram of the spleen from the infected guinea pigs, and the minimum infecting dose (MID) for guinea pigs were tested.【Result】B. abortus 343 is a smooth-type, Gram-negative, Koch's staining red strain, which can reflect glaucous radiance from its surface culture. It releases H2S during its metabolism, and grows on TSA containing thionine or alkaline azaleine, and the growth of which was not dependent on CO2. The antigen prepared from B. abortus 343 was positive with A-type serum in plate agglutination test, and it could induce the immunized mice to produce the specific antibody. The bacteria were isolated from the spleen of the tested mice 29 weeks post infection with a dose of 1×105 CFU. While infected with a dose of 1×109 CFU to the female guinea pigs, 2.4×105 CFU - 1.2×106 CFU could be cultured from spleen per gram 14 days post infection. It could induce specific smooth-type antibody from all guinea pigs infected with a dose of 1×105 CFU, and 1 month post infection the titers of serum were high from 320 to 1 280 in agglutination test. To guinea pigs, the MID is about 40 CFU. 【Conclusion】A middle virulence B. abortus strain, which can be used as a reference strain in the further study was identified. As a new isolate, the detailed identified B. abortus 343 strain is a good supplement to the brucella resources.

Identification, Phylogenetic and Tissue Expression Microarray Analyses of the Sirtuin Gene Family in Silkworm (Bombyx mori)

CHEN Cong, SONG Jiang-Bo, MENG Gang, TONG Xiao-Ling, DAI Fang-Yin, LU Cheng

Scientia Agricultura Sinica. 2014, 47(13): 2659-2670. doi:10.3864/j.issn.0578-1752.2014.13.018

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【Objective】The objectives of this study are to identify sirtuin genes from silkworm genome, analyze the gene structure, protein structure, physicochemical properties, phylogeny and tissue expression pattern, and further to provide a basis for the function of sirtuins of silkworm and silkworm model organismal system.【Method】Based on silkworm genome database and bioinformatic method, silkworm sirtuin family genes were identified by comparative genomic analysis. The opening reading frame (ORF) of sirtuins was predicted by ORFfinder, exon and intron were predicted by SIM4. Gene structure was created using GSDS. Physicochemical properties of sirtuin proteins were done by ExPaSy server, multiple sequence alignment and its secondary structure analysis were done by CLUSTAL_X and ESpript, protein structure and phylogenetic tree were analyzed using SMART and MEGA5.0 software. The expression pattern of sirtuin genes in different tissues was analyzed based on the existing microarray database of 3-day-old 5th instar larvae. The expression level of sirtuin genes of silkworm in different tissues of 3-day-old 5th instar larvae were determined by quantitative RT-PCR.【Result】A total of 5 sirtuin genes (Bmsirt2, Bmsirt4, Bmsirt5, Bmsirt6, Bmsirt7) were systematically identified from silkworm and classified into 4 classes (I, II, III, IV). These genes were distributed on 5 chromosomes, and they were single copy genes. Homologous alignment and phylogenetic analysis revealed that the sirtuin genes in silkworm had apparent orthologous relationship with homologous genes from other insects respectively and they were highly homologous, also lack of sirt3. Protein structures showed that sirt6 had two sir2 domain associated together, as the same as other organisms. Microarray analysis showed that the sirtuin family genes had transcriptional active in multiple tissues. Analysis by real time quantitative PCR indicated that Bmsirt4 was expressed relatively lower abundance in fatboby and silk gland. Bmsirt5 was expressed relatively higher abundance in testis and midgut, but was lower abundance in fatboby, hemocyte, and silk gland. This was consistent with the microarray data. 【Conclusion】 Five genes of sirtuin family in silkworm were identified by genome-wide screening, they were classified into 4 classes, highly homologous with other insects. Microarray data was consistent with the result of real-time fluorescence quantitative PCR, and the analysis showed that the tissue expression pattern was diversity.

Validation and Application of Function Markers CWI22 and CWI21 of TaCwi-A1 Gene Related to Wheat Kernel Weight

XIANG Ji-Shan-1, 2 , MU Pei-Yuan-1, 2 , SANG Wei-1, 2 , NIE Ying-Bin-1, XU Hong-Jun-1, ZHUANG Li-2, 3 , CUI Feng-Juan-1, 2 , HAN Xin-Nian-1, ZOU Bo-1

Scientia Agricultura Sinica. 2014, 47(13): 2671-2679. doi:10.3864/j.issn.0578-1752.2014.13.019

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【Objective】In order to provide some information for molecular marker assisted selection, the function markers CWI22 and CWI21 of TaCwi-A1 gene related to wheat kernel weight were validated. The allelic frequencies at TaCwi-A1 locus were detected by those markers in Xinjiang wheat germplasm. 【Method】The 110 Xinjiang winter wheat varieties were genotyped with CWI22 and CWI21, and the difference in thousand kernel weight (TKW) among varieties with TaCwi-A1a and TaCwi-A1b were analyzed. The allelic variations at TaCwi-A1 locus were detected by CWI22 and CWI21 in a total of 1 241 Xinjiang wheat germplasms. 【Result】In the 110 Xinjiang winter wheat varieties, a 402-bp fragment could be amplified by CWI22 in 46 varieties, which means they had genotype of TaCwi-A1a, a 404-bp fragment could be amplified by CWI21 in 64 varieties, which means they had genotype of TaCwi-A1b. And the TKW of varieties with TaCwi-A1a (43.5 g) was significantly higher than that with TaCwi-A1b (40.9 g) (P＜0.05). In the 1 241 Xinjiang wheat germplasms, the frequencies of TaCwi-A1a and TaCwi-A1b were 62.6% and 37.4% in all germplasms, 63.0% and 37.0% in winter wheats varieties, 61.7% and 38.3% in spring wheat varieties, respectively. In different types of varieties of both winter and spring wheat germplasm, the frequency of TaCwi-A1a was in order of exotic varieties (lines)＞domestic varieties (lines)＞breeding varieties (lines)＞released varieties＞landraces. In Xinjiang released varieties, the frequencies of TaCwi-A1a and TaCwi-A1b were 40.0% and 60.0% in winter wheats, 68.6% and 31.4% in spring wheats, respectively. In three stages of before 1990, 1991 to 2000, and after 2001, the frequencies of TaCwi-A1a and TaCwi-A1b were 11.1% and 88.9%, 50.0% and 50.0%, 69.2% and 30.8%, respectively. 【Conclusion】The markers CWI22 and CWI21 of TaCwi-A1 gene can efficiently distinguish the differences of wheat TKW, which can be used for marker assisted selection of kernel weight (KW). In Xinjiang wheat germplasm, TaCwi-A1a gene has a high frequency. And the frequency of TaCwi-A1a gene in winter wheat germplasm is a little higher than spring wheat germplasm, introduced varieties (lines) is higher than breeding varieties (lines), breeding varieties (lines) is higher than landraces. In both landraces and breeding varieties (lines), the frequencies of TaCwi-A1a gene in spring wheat varieties are higher than winter wheat varieties, which means that the breeding selection of KW in winter and spring wheat are different in Xinjiang, but there had high selection pressure in all, which makes the frequency of TaCwi-A1a gene in released varieties increase.

QTL Mapping and Interaction Analysis of Seed Protein Content and Oil Content in Soybean

HOU Meng-1, QI Zhao-Ming-1, HAN Xue-2, XIN Da-Wei-1, JIANG Hong-Wei-2, LIU Chun-Yan-2, WU Qiong-1, SUI Li-Li-4, HU Guo-Hua-2, 3 , CHEN Qing-Shan-1, 3

Scientia Agricultura Sinica. 2014, 47(13): 2680-2689. doi:10.3864/j.issn.0578-1752.2014.13.020

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【Objective】 Quantitative trait loci associated with protein and oil contents were identified and epistatic interactions in soybean. The results will offer a clue for quality gene mining and molecular breeding in soybean. 【Method】 Total 147 recombination inbred lines (RIL) were derived from a cross of Charleston and Dongnong 594, the F2:19-F2:20 generation of RIL was used as experimental materials. Using CIM and MIM model method by Windows QTL Cartographer V.2.5, QTL associated with protein and oil contents were identified. The epistatic effect and environmental effect between QTLs were analyzed by QTL Network 2.1. 【Result】 Under six planting environments including Harbin, Hongxinglong, Jiamusi, and Mudanjiang in 2011 and 2012, a total of nine QTLs for protein content and eleven QTLs for oil content were mapped. Protein content QTLs were mapped on six linkage groups A1, C2, D1a, G, H, and O. The QTLs explained 5.3%-18.6% of phenotypic variation, the maximum rate of qPro-H-1 on linkage group H was 18.6%, the minimum rate of qPro-D1a-2 on linkage group D1a was 5.3%. Five protein content QTLs in single planting environment were simultaneously detected by two methods, which were qPro-O-1, qPro-A1-1, qPro-D1a-1, qPro-D1a-2, and qPro-C2-2. Oil content QTL were mapped on eight groups A1, A2, B1, C2, D1a, E, L, and M. The QTLs explained 7.1%-24.4% of phenotypic variation, the maximum rate of qOil-B1-2 on linkage group B1 was 24.4%, the minimum rate of qOil-C2-3 on linkage group C2 was 7.1%. Two oil content QTLs qOil-C2-1, qOil-M-1 were detected in single planting environment. Besides, two oil content QTLs were detected in over 2 environments, qOil-A1-1 was indentified in 2011 in Harbin and in 2011 in Hongxinglong two planting environments, qOil-B1-2 was indentified in 2011 in Hongxinglong, in 2011 in Mudanjiang and in 2012 in Harbin three planting environments. A total of three pairs protein epistatic effect QTL and four pairs oil content epistatic QTL were found. Protein epistatic effect was 0.2068-0.3124, the QTLs explained 0.0227%-0.0265% of phenotypic variation, were mapping on linkage groups A1, C2, D1, and E, qPro-A1-3 and qPro-C2-1 were negative interaction effect, others were positive interaction effect. Both two epistatic effect QTLs on linkage groups A1 and D1a. Oil epistatic effect was between 0.0926 and 0.1682, the QTLs explained 0.0294%-0.0754% of phenotypic variation, and the QTLs were mapping on linkage groups A1, C2, I, J, N and O. qOil-C2-4 and qOil-N-1 had negative interaction effect, and the other 3 loci had positive interaction effect to phenotype. Interactions were identified between qOil-N-1 with qOil-C2-1 and qOil-N-1 with qOil-C2-4, respectively. Six pairs of QE interaction effects QTLs were detected, qPro-D1a-3 and qPro-E-1 pairs were not detected in Jiamusi in 2012. 【Conclusion】 In total, nine QTLs related to protein content and eleven QTLs related to oil content were mapped. Three QTL pairs with epistatic effects for protein content and four QTL pairs with epistatic effects for oil content were detected, respectively.

Cloning and Bioinformatics Analysis on CDS of CYGB Gene in Yak

SUN Xue-Jing-1, 3 , DU Xiao-Hua-1, 3 , YANG Xiao-Pu-1, LUO Yu-Zhu-3, LIU Xia-2, 3

Scientia Agricultura Sinica. 2014, 47(13): 2690-2698. doi:10.3864/j.issn.0578-1752.2014.13.021

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【Objective】In order to enrich basic data in yak CYGB gene, CDS region of yak CYGB gene was cloned and analyzed by bioinformatics method. 【Method】 Total RNA of yak hippocampus tissue was extracted and reverse transcribed into cDNA by RT-PCR technology. Specific primers were designed according to cDNA sequence of cattle CYGB gene in the GenBank (GenBank accession No.：DV874786.1) by online software Primer 3.0. The CDS region and part of 5′ UTR and 3′ UTR in yak CYGB gene were cloned from yak hippocampus total RNA by PCR amplification, TA cloning and nucleic acid sequencing technology. The primary structure, secondary structure, tertiary structure, physicochemical properties, homology were analyzed and phylogenetic tree of CYGB was constructed by online software like ProtParam, PredictProtein, SWISS-MODEL and Lasergene7.1 software package. The three-dimensional structure was modified and output by PyMol software. The protein subcellular localization was predicted by online subcellular localization tool PSORT II Prediction, and the protein function was predicted by Protfun software.【Result】The 650 bp length CYGB gene in yak was got by cloning, including the 573 bp length CDS region (GenBank accession No.：KF669898), and the bases composition were 20.59%A, 16.40%T, 33.33%G, 29.67%C, encoding 190 amino acids. Alignment with CDS and amino acid sequence of cattle CYGB gene, four base mutations were found and amino acid was not mutated, four mutations are synonymous mutations. The formula of protein encoded by CYGB gene in yak was C964H1513N263O278S7, and the molecular weight was 21.5 kD, the theory isoelectric point was 6.32, the extinction coefficient was 24075, the instability index was 48.43, the aliphatic index was 83.63, and the grand average of hydropathicity was -0.301. It was an unstable and soluble protein. Its estimated half-life is 30 hours in mammal reticulocyte. The secondary structure of CYGB was mainly α-helices and random coil, α-helices was 64.21% and random coil was 35.79%, CYGB was an α class protein. The tertiary structure of CYGB was a “three-over-three” α-helical sandwich structure. Subcellular localization of CYGB was in the cytoplasm (65.2%), nucleus (17.4%), mitochondria (13.0%) and vesicles of secretory system (4.3%), mainly in the cytoplasm, which CYGB may play a role of signal transducer or transcription regulation in the energy metabolism and cofactor biosynthesis. The amino acid homology of CYGB compared yak to cattle, sheep, dog, mouse, rat, chicken, monkey, chimpanzee and human were 100%，98.9%，97.8%，95.3%，93.7%，78.8%，98.4%，95.8% and 96.8%, respectively. There was a high homology among different species, and phyletic evolution was the same as their genetic relationship. The research indicated that the CYGB gene coding region was conservative in the course of evolution.【Conclusion】The 573 bp length CDS region of yak CYGB gene was got by RT-PCR, TA cloning and nucleic acid sequencing technology for the first time, and the nucleotide sequence, the encoded amino acid sequence and protein structure and function were analyzed. CYGB in yak was a soluble and acidic protein that 190 amino acid residues composed, which plays an important role in the energy metabolism and cofactor biosynthesis. CYGB gene coding region was conservative in the course of long-term biological evolution. The gene was cloned and analyzed, thus providing a theoretical basis for revealing the genetic characteristics of yak CYGB gene.

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