Table of Content

01 April 2016, Volume 49 Issue 7

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CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS

The Development of CRISPR/Cas9 System and Its Application in Crop Genome Editing

JING Run-chun, LU Hong

Scientia Agricultura Sinica. 2016, 49(7):  1219-1229.  doi:10.3864/j.issn.0578-1752.2016.07.001

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In this review, the steps of development of the CRISPR/Cas9 genome editing system and its applications in various plant genomes were highlighted. The CRISPR/Cas9 genome editing technology originates from the prokaryotic adaptive immune systems that confer resistance to foreign genetic elements such as plasmids and phages. The natural CRISPR/Cas systems show extensive structural and functional diversity. Based on the Cas protein contents and amino acid sequences, the natural CRISPR/Cas systems have been classified into three major classes, Type I, TypeⅡ and Type III. The TypeⅡCRISPR/Cas system is the engineered one for targeted genome editing purpose in most of cases so far, as it needs optimization of the Cas expression and design of the sgRNA only. In 2013, the first applications of CRISPR/Cas9 genome editing technology in plants were published. Since then, the CRISPR/Cas9 system has been used in various plant species for targeted genome editing. Like ZFNs and TALENs, CRISPR/Cas9 system uses engineered nuclease to generate double-strand breaks (DSBs) on the targeted DNA site, and subsequently to stimulate cellular DNA repair mechanisms by exploiting either NHEJ pathway or HDR pathway to generate small insertions/deletions/genome modifications. CRISPR/Cas9 technology allows researchers to perform targeted mutagenesis on target genes of different crops, precisely and easily changing the sequences and functions of particular genes at exact chromosomal locations in different plant genomes. Compared with ZFNs and TALENs technologies, CRISPR/Cas9 genome editing system is based on RNA-guided engineered nucleases, and is easier to manipulate. Furthermore, CRISPR/Cas9 is capable of introducing DSBs at multiple sites. The potential of multiplexing provides practical advantages over ZFNs and TALENs technologies, to edit multiple target genes in the same pathway simultaneously. Due to the practical advantages of CRISPR/Cas9 over the other genome editing technologies, it establishes a prosperous outlook in gene discovery and trait development in crop genetic improvement and breeding studies. In this review, the possible applications of CRISPR/Cas9 genome editing technique in various aspects of plant genetics and breeding were also discussed, except the targeted genome editing. CRISPR/Cas9 genome editing technology is another stepping stone in utilizing genetic manipulation in genetic studies and breeding, after genetic modification. Unlike genetic modification, CRISPR/Cas9 genome editing technique generates phenotypic variation that is indistinguishable from that obtained through natural means or conventional mutagenesis. This ambiguity challenges the current GMO regulatory definitions, and provides a potential barrier for further use of CRISPR/Cas9 genome editing technique in crop genetics and breeding.

Progresses and Perspective of the Function of MYB Transcription Factor MIXTA and Its Orthologous Gene

ZHANG Yang, WU Jia-yan, WU Ya-ni, TANG Ming-wei, XIE Li-nan

Scientia Agricultura Sinica. 2016, 49(7):  1230-1241.  doi:10.3864/j.issn.0578-1752.2016.07.002

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Nowadays, the morphology of epidermal cells in the angiospermaes is a universal biological phenomena, and it roughly includes the shape of the epidermal cells, the formation of epidermal cells attached derivatives, and the differentiation of trichomes and root-hair cells, etc.. Relying on large amounts of experimental results, people have found that the biological functions of MIXTA and its orthologous genes have these related phenomena. MIXTA encodes a MYB-like transcription factor with R2R3 domain. It got widespread attention since 1994 when MIXTA had been firstly reported in the Antirrhinum majus,and also got more in-depth studies and discussions in different species. It was found that MIXTA promoted the petals epidermal cells changed to be conical and made the petals inner epidermal cells have the velvet-like texture, on the contrary petals epidermal cells changed to flattening and the velvet-like texture in petals within epidemals disappeared when MIXTA gene had mutations in the wild type of Antirrhinum majus. Ectopic expression of MIXTA regulated the growth of the cone cells and multicellular glandular trichomes in different tissues. During the long-term study, some MIXTA homologous genes have been found in many other species except A. majus,collectively known as MIXTA-like, such as Thalictrum thalictroides, Erythranthe lewisii, Solanum lycopersicum, Arabidopsis thaliana,etc. especially in Arabidopsis thaliana. The functions of these genes in Arabidopsis thaliana were similar to MIXTA in Antirrhinum majus and research on their downstream genes regulating pathways were more clearly. At the same time, MIXTA, as a MYB-like transcription factor, however, could be through the interactions with bHLH transcription factors and WD40 transcription factor to indirectly control the synthesis of anthocyanin. MIXTA affects a series of biological characteristics, such as the surface temperature of the petals, smell, color, moisture, texture and optical properties. It also indirectly influenced plant pollination by changing the phenotypes of the plant petals and established their own defense systems. The biological functions of MIXTA are so extensive that studying it had an important biological significance. In this article, the basic structure of MIXTA gene was first briefly introduced, and using bioinformatics technology including the alignment of amino acid sequences and phylogenetic analysis to analyze the relationship between MIXTA and its orthologous proteins and R2R3 domain that they all had. After that, from three aspects including the shape of epidermal cells (turning to be conical), the formation of the affiliated derivatives on epidermis cells (including cuticular wax biosynthesis, trichomes and the differentiation of the root-hair cells), participating in hormone metabolic pathways to indirectly regulate the epidermal cells morphology to discuss the specific function of MIXTA and its homologous genes. Finally, the future of MIXTA application in agriculture, ornamental horticulture was discussed which will provide a reference for researchers in this field.

Cloning and Expression Analysis of Argininosuccinate Synthetase Gene GhASS1 from Gossypium hirsutum

WANG Hui-fei, SUN Yan-xiang, FENG Xue, ZHANG Yi-ming, YANG Jiang-tao, MA Mei-fang, CHEN Guang

Scientia Agricultura Sinica. 2016, 49(7):  1242-1253.  doi:10.3864/j.issn.0578-1752.2016.07.003

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【Objective】This research was conducted to clone argininosuccinate synthetase gene from Gossypium hirsutum, obtain the fusion protein by prokaryotic expression system, detect the argininosuccinate synthetase activity of the fusion protein, and identify its effects on the growth, salt tolerance, L-citrulline (L-Cit) and L-arginine (L-Arg) content of engineering bacteria, aiming to lay a foundation for this gene’s function and mechanism. 【Method】The homologous cDNA fragment, named as GhASS1, was obtained from young leaves of cotton by the querying probe, a putative argininosuccinate synthetase cDNA sequence from Arabidopsis thaliana, in silico cloning and RT-PCR reaction. This fragment’s information was acquired by T/A cloning and sequencing. The genomic DNA and putative protein structure, function domain and homology were analyzed by bioinformatics software, and the phylogenetic tree was built. Its expression responses to salt tolerance were investigated by qRT-PCR. After the open reading frame of GhASS1 was linked to pCold-TF, the fusion expression vector, pCold-GhASS1, was constructed, and then transformed into the bacterial strain of Rosetta (DE3) plysS for expressing the recombination protein under the induction of IPTG. The molecular weight of recombination protein was tested by SDS-PAGE. The enzyme activities and specific activities were determinated by the method of pyrophosphoric fluorescence. The contents of free L-Arg and L-Cit in pCold-GhASS1 engineering bacteria were assayed by HPLC. The growth status of engineering bacteria was monitored under different NaCl concentrations in culture medium and different growth temperatures. The contents of free L-Arg and L-Cit, L-Arg/L-Cit ratio were compared. 【Result】 Sequencing analysis showed the genomic DNA of GhASS1 had 10 exons and 9 introns which was located on chromosome 1 of D genome in cotton, its cDNA fragment length was 1 584 bp containing 5′-untranslated region of 22 bp, the open reading frame of 1 485 bp and 3′- untranslated region of 77 bp, encoding 494 amino acid residues with a molecular mass of 54 kD and isoelectric point of 6.74. Sequence alignment analysis showed that GhASS1 shared 21.81%, 41.1% and 78.5% identity with the homologues from E. coli, yeast and Arabidopsis thaliana, respectively, and had the typical function domains of argininosuccinate synthetase. Phylogenetic analysis showed that GhASS1 was most closely related to the homologues from tomato, potato and tobacco, while was farthest to those from Picea sitchensis and Selaginellae moellendorfii. The expression of GhASS1 was up-regulated to the highest after 1 d under salt stress, then gradually declined in the leaves, indicating GhASS1 mRNA took part in the early response to salt tolerance. The SDS-PAGE analysis revealed that the fusion recombinant protein of GhASS1 was a molecular weight of 108 kD as expected. The fusion protein had argininosuccinate synthase activity in vitro. Compared with the control bacteria with pCold-TF, the contents of free L-Cit were decreased, while those of free L-Arg were increased in the engineering bacteria with pCold-GhASS1. In addition, the engineering bacteria with pCold-GhASS1 could be earlier into the logarithmic growth than the control, also showed stronger growth vigor and higher free L-Arg/L-Cit ratio under high NaCl contents in LB medium, revealing the engineering bacteria had stronger metabolic flow from L-Cit to L-Arg than the control. 【Conclusion】 It was the first report on the identification of argininosuccinate synthetase gene from cotton in plant. Its catalysis and physiology function were analyzed by the prokaryotic expression system, suggesting that it could be involved in the regulation of growth and ability of salt resistance in plant.

TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY

Effects of Nitrogen Fertilization Managements with Residues Incorporation on Crops Yield and Nutrients Uptake Under Different Paddy-Upland Rotation Systems

ZHANG Wei-le, DAI Zhi-gang, REN Tao, ZHOU Xian-zhu, WANG Zhong-liang, LI Xiao-kun, CONG Ri-huan

Scientia Agricultura Sinica. 2016, 49(7):  1254-1266.  doi:10.3864/j.issn.0578-1752.2016.07.004

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【Objective】The effects of nitrogen (N) fertilization management on crop yield, total N accumulation and partial factor productivity were studied under the condition of returning residues in different paddy-upland rotations. 【Method】Field experiments were conducted in 14 counties (e.g., Xiaonan, Songzi, Yingcheng) of Hubei Province under rice-oilseed rape and rice-wheat rotation systems in the period of 2013-2014. Five treatments were followed as: 1) Conventional N fertilization in 3  times, 2) conventional N fertilization in 3 times with residues incorporation, 3) high N fertilization rate in 3 times with residues incorporation, 4) N fertilization in 2 times, and 5) N fertilization in 2 times with residues incorporation. Crop yields (rice, rapeseed and wheat), total N accumulation and partial factor productivity were analyzed among the treatments.【Result】 Crop yield, aboveground biomass and total N accumulation were not affected by high N application rate under residue incorporation in the rice-oilseed rape rotation but were significantly increased in the rice-wheat rotation. Compared with conventional N fertilization in 3 times for the rice-wheat cropping rotation, high N fertilization rate in 3 times with residues incorporation rice and wheat yield increments were 0.632 and 0.564 t·hm^-2 on average, with the increasing rates of 6.85% and 10.67%, respectively. Aboveground biomass increments were 1.50 and 1.07 t·hm^-2 on average, with increasing rates of 8.11% and 9.06%, respectively. Total N accumulation increments were 11.54 and 23.57 kg·hm^-2 on average, with increasing rates of 7.88% and 21.28%, respectively. Total N accumulation of rice and wheat increment was 35.11 kg·hm^-2, with increasing rate of 13.65%. N application at 2 times with residues incorporation would satisfy crop yield and total N accumulation compared with conventional N fertilization at 3 times. Specifically for the rice-wheat rotations, rice and wheat yield increments were 0.439 and 0.385 t·hm^-2 on average, with increasing rates of 5.12% and 7.63%, respectively. Total N accumulation increments were 11.09 and 21.06 kg·hm^-2 on average, with increasing rates of 8.26% and 20.82%, respectively. Total N accumulation increment was 32.14 kg·hm^-2 for the rice-wheat cropping rotation, with increasing rate of 13.66%. For N efficiency, regular application rate of N obtained high partial factor productivity of applied N (PFP_N, averaged values of PFP_N were 52.03 to 59.29 kg·kg^-1 for rice, 10.62 to 11.12 kg·kg^-1 for oilseed rape, and 33.63 to 36.20 kg·kg^-1 for wheat), partial factor productivity of applied N (PFP_N) with residues incorporation was better than that without residues incorporation for equal nitrogen rates, especially when we moved N fertilizer forward with residues returning. For rice-oilseed rape rotation system, Compared with conventional N fertilization at 3 times and N fertilization in 2 times, conventional N fertilization in 3 times with residues incorporation and N fertilization in 2 times with residues incorporation the PFP_N of rice increments were 2.45 and 4.07 kg·kg^-1 on average, with increasing rates of 4.36% and 7.37%, respectively. Oilseed rape increments were 0.36 and 0.49 kg·kg^-1 on average, with increasing rates of 3.38% and 4.62%, respectively. For rice-wheat rotation system, rice increments were 3.88 and 1.64 kg·kg^-1 on average, with increasing rates of 7.46% and 3.10%, respectively. Wheat increments were 1.60 and 1.93 kg·kg^-1 on average, with increasing rates of 4.75% and 5.65%, respectively. Compared with conventional N fertilization at 3 times, the PFP_N values of rice and oilseed rape would increase 5.68% and 4.00% under N Fertilization at 2 times with residues incorporation, respectively. For the rice-wheat rotation system, N application at 2 times with residues incorporation would increase the PFP_N values by 5.12% and 7.63% for rice and wheat, respectively.【Conclusion】With residues returning, moving fertilizer-nitrogen forward would achieve high and stable crop yield and enhance N efficiency under different paddy-upland rotations.

Effects of Plant Growth Regulators S3307 and DTA-6 on Physiological Metabolism and GmAC Gene Expression in Soybean

SUN Fu-dong, FENG Nai-jie, ZHENG Dian-feng, CUI Hong-qiu, LIU Chun-juan, HE Tian-ming, ZHAO Jing-jing

Scientia Agricultura Sinica. 2016, 49(7):  1267-1276.  doi:10.3864/j.issn.0578-1752.2016.07.005

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【Objective】 Pods are important source organs for temporary reserve photosynthate in soybean grain growth progress. It is a key factor of pods abscission to restrict soybean production. In order to investigate the internal mechanism of chemical control technology to improve soybean yield, this study used two plant growth regulators to discover the different effects on physiological metabolism in pods and the expression of abscission cellulose enzyme gene (GmAC) in the abscission zone. This study will provide a theoretical basis for high-yield, high-quality and high-efficiency in soybean production.【Method】Field experiments were conducted with soybean Kangxian 6. We sprayed growth promoter regulator 2-N,N-diethylamino ethyl caproate (DTA-6) 60 mg·L^-1 and the retardant regulator uniconazole (S3307) 50 mg·L^-1 or water during R1 growth stage in 2012 and 2013. The pods of each treatment were used to examine pod physiological indicators including soluble sugar, sucrose, starch, MDA, POD and AC on 35, 42, 49 and 56 days after spraying in each treatment. Abscission zone tissues between stem and soybean pods were cut on the 5th day after treatment, to determinate the expression of cellulose enzyme gene (GmAC) using applied RT-PCR method. 【Result】(1) Compared with the control, DTA-6 and S3307 treatments had significantly lower soluble sugar, except on 35 d after spraying where the DTA-6 treatment sugar content was higher. The content of sucrose in pods decreased after spraying DTA-6 and S3307 from the 35 d to the 49 d, and increased on the 56 d after spraying. Starch increased in most of treatment-spray time conditions except in the S3307 treatment on the 42 d after spraying. (2) The content of MDA decreased after 35, 49 and 56 d after spraying. The activity of POD increased after 35 d, 42 d and 56 d after spraying. The activity of AC decreased on the 35 d, 49 d and 56 d after spraying S3307 and decreased from 42 d to 49 d after spraying DTA-6. (3) The relative expression of GmAC in abscission zone after spraying DTA-6 was down-regulated, but the relative expression after spraying of S3307 was up-regulated. (4) DTA-6 and S3307 treatments effectively improved yield properties such as the number of pods per plant, grains per pod and one-hundred grain weight, compared with control. The yield improved by 6.2% and 1.4% in 2012, and 4.8% and 5.3% in 2013. 【Conclusion】Our observations suggest that DTA-6 and S3307 increased transportation and accumulation of assimilate in pods, enhanced the antioxidant enzyme, reduced membrane lipid peroxidation products, decreased the key enzyme activity of abscission pods, benefited the pods, and ultimately improved the production of soybean. The relative expression of GmAC was down-regulated in the treatment with DTA-6, and DTA-6 treatment was better.

PLANT PROTECTION

Expression and Functional Analysis of CsWRKY30 in Cucumber Under Propamocarb Stress

LI Sheng-nan, QIN Zhi-wei, XIN Ming, ZHOU Xiu-yan

Scientia Agricultura Sinica. 2016, 49(7):  1277-1288.  doi:10.3864/j.issn.0578-1752.2016.07.006

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【Objective】The objective of this study is to screen, clone and analyze the function of the cucumber transcription factor gene CsWRKY30, which was up-regulated under propamocarb stress in previous transcriptome analyses, CsWRKY30, and to understand the molecular mechanism of low propamocarb residue in cucumbers.【Method】The full length of CsWRKY30 was cloned by PCR, and protein sequences and promoter region of the gene were analyzed with NCBI and PlantCARE separately. The real-time PCR was used to analyze the expression pattern of CsWRKY30 under different treatments. The subcellular localization of CsWRKY30 was conducted through fusion with GFP protein. CsWRKY30 was over-expressed in Arabidopsis by the inflorescence infection method and got pure transgenic lines to identify gene function.【Result】The full length of CsWRKY30 was 1 014 bp and encoded 337 amino acids, which contained a WRKY conservative domain consisting of 60 amino acids. Expression pattern analysis showed that CsWRKY30 was induced by propamocarb stress in the low propamocarb residue D0351, while there was no differences in the high propamocarb residue D9320. The expression level of CsWRKY30 in D0351 was significantly higher than that in the control during 0.5-9 h, while the expression of this gene was no longer significantly up-regulated after 24 h. Tissue specific expression analysis showed that CsWRKY30 was mainly expressed in cucumber fruits. Subcellular localization indicated that CsWRKY30 was mainly located on the nucleus in plant cells. Over-expression CsWRKY30 Arabidopsis under propamocarb stress test showed that there were no significant phenotypic differences between transgenic Arabidopsis and wild types under the untreated condition, while under 2 mmol?L^-1 propamocarb stress the transgenic Arabidopsis germination rate and root length were apparently higher than that of the wild type. Expression of CsWRKY30 also showed a trend of increasing under Corynespora cassiicola treatment and was induced by abscisic acid (ABA), while showing no effects in drought and high salt stresses.【Conclusion】CsWRKY30 played an important role under propamocarb stress in cucumber, and over-expression of CsWRKY30 enhanced transgenic plants tolerance.

Residue and Dietary Exposure Risk Assessment of Four Pesticides in Apple

YE Meng-liang, NIE Ji-yun, XU Guo-feng, YAN Zhen, ZHENG Li-jing

Scientia Agricultura Sinica. 2016, 49(7):  1289-1302.  doi:10.3864/j.issn.0578-1752.2016.07.007

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【Objective】 The objectives of this study are to detect pesticide (carbendazim, thiophanate-methyl, imidacloprid and chlorbenzuron) residues in apples from China and evaluate their dietary exposure, identify and quantify the risk levels of dietary intake of the 4 commonly used pesticides in China, and to provide evidence for safe production, consumption, and safety supervision of apple. 【Method】 Based on the 282 apple samples from two main apple producing areas, including the Bohai bay (Liaoning, Shandong and Hebei) and the Northwest loess plateau (Shaanxi, Shanxi and Henan), professional risk assessment software @Risk was used to evaluate the dietary intake (exposure) risk of apple with the 4 pesticides by building a nonparametric probabilistic evaluation model. First, data on the 4 pesticides residual content in 282 apple samples were collected for distribution fitting, which were checked by three statistical test methods: the Chi-Squared test, the Anderson-Darling test and the Kolmogorov-Smirnov test, and the optimum fitting distribution was selected from the results. The supervised trials median residue (STMR) and highest residue (HR) values were superseded by the values of optimum distribution fitting. %ADI and %ARfD indicate the chronic dietary intake risk and acute dietary intake risk, respectively. 【Result】 Of the 282 apple samples tested, 90.4% of apple samples (255) had detectable residues. Of the 4 common used pesticides, the detectable rate of carbendazim (CA) was 81.9%, which was the most frequently detectable pesticide, followed by thiophanate-methyl (TH) (52.1%), imidacloprid (IM) (39.0%), and chlorbenzuron (CH) (31.2%). The vast majority of samples pesticide residues were at lower levels, and the highest concentration was 0.9251 mg·kg^-1 (CA), which was still lower than the MRL (3.0 mg·kg^-1). The mean residual concentrations of the 4 pesticides in samples were in the following order: CA (0.1042 mg·kg^-1) ＞CH (0.0182 mg·kg^-1) ＞TH (0.0082 mg·kg^-1) ＞IM (0.0050 mg·kg^-1). There were differences in residual concentrations’ dispersion degree, and the variance coefficient was up to 136.4% (CA), 232.8% (TH), 214.8% (IM) and 174.1% (CH), respectively. The distribution law of the residual concentration of the 282 apple samples was obvious, the proportion of samples was reduced gradually in a trend with the increase of residual concentration. Twenty-seven samples (9.6% of all tested samples) were residue-free of the 4 pesticides, 198 samples (70.2%) had more than two residues, and 19 samples (6.7%) even had 4 kinds of pesticide residues. The chronic dietary intake risks for different age groups consuming apples with these 4 pesticides were 0.2120%-35.1100% (CA), 0.0051%-0.8240% (IM), 0.0049%-0.1710% (TH) and 0.0004%-0.0152% (CH), respectively. The acute dietary intake risks were 0.1940%-16.0500% (CA) and 0.0122%-0.9400% (IM), respectively. The infants (aged 2 to 6) and children (aged 7 to 13) were significantly higher than that of other groups, because of higher apple intake and lower body weight, so that they were the vital monitoring objects. The pesticide intake risks were declined gradually with the age growth, and the risks were higher with the larger percentile values selected.【Conclusion】CA, TH, IM and CH residues in Chinese apples had a relatively high detectable rate. Nevertheless, the residual concentrations of all the samples tested were under MRLs. Both chronic and acute dietary intake risks for the general population consuming apples with the selected 4 pesticides were low. The infants and children were significantly higher than that of other groups and should be paid more attention.

SOIL & FERTILIZER·WATER-SAVING IRRIGATION·AGROECOLOGY & ENVIRONMENT

Trend Analysis and Estimation of Net Primary Productivity in Yinchuan Basin

LI Bo-yan, REN Zhi-yuan

Scientia Agricultura Sinica. 2016, 49(7):  1303-1314.  doi:10.3864/j.issn.0578-1752.2016.07.008

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【Objective】The objective of this study is to simulate the net primary productivity (NPP) using the NPP model in Yinchuan Basin based on the remote sensing data and meteorological data from 2000 to 2010. The spatio-temporal changing process of NPP and its trend was analyzed (monthly and yearly), and the Yinchuan Basin’s vegetation NPP spatio-temporal pattern and variation were described for discussing the coupling relationship between the NPP and vegetation health index (VHI).【Method】Using SPOT VEGETATION data (1 km spatial resolution) from 2000-2010, as well as NDVI data and metrological data from 2000-2010, the NPP model was applied to analyze the spatial-temporal characteristics of NPP changes in Yinchuan Basin. The estimation was based on CASA model, linier regression equation and SVD method. 【Result】 Analysis of the spatial-temporal pattern of NPP showed that the NPP in Yinchuan Basin was increasing slowly at the rate of 0.51 per month, the most in July and October. The average annual change was 0.24, corresponding with the changes of rainfall. The NPP in Yinchuan Basin was higher in the south and east, while lower in the middle and west. The lowest value lies in Xixia region, Xingqing region and Jinfeng region: Dawukou District＞Yongning County＞Lingwu City＞Xixia District＞Qingtongxia City＞Litong District＞Pingluo County＞Helan County＞Huinong District＞Xingqing District＞Jinfeng District. The trend of NPP had a negative relationship with the amount of towns and the number of population. The correlation of the first mode was -0.69, showing the VHI made a negative influence on NPP. 【Conclusion】The estimated regional CASA model was precise to the actual data. The NPP in Yinchuan Basin was generally benign except some areas. Soil moisture can affect the NPP showing that drought can be predicted by using the value of NPP.

Responses of Soil Microbial Biomass and Soluble Organic Matter to Different Application Rates of N：A Comparison Between Liaochun 10 and Liaochun 18

YANG Xin-yi, LIU Xiao-hu, HAN Xiao-ri, DUAN Peng-peng, ZHU Yu-cui, QI Wen

Scientia Agricultura Sinica. 2016, 49(7):  1315-1324.  doi:10.3864/j.issn.0578-1752.2016.07.009

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【Objective】 Liaochun10 and Liaochun18 are two of the predominant wheat varieties in Northeast China. However, the difference of the two wheat varieties in sustaining soil quality is still not well understood. It is worth to study the responses and transformation of soil microbial biomass C and N (SMBC, SMBN), soil soluble organic C and N (SSOC, SSON) to different application rates of N. 【Method】 A pot experiment was conducted with different wheat varieties and six treatments were designed, i.e., N0: 0 kg·hm^-2, N1: 45 kg·hm^-2, N2: 90 kg·hm^-2, N3: 135 kg·hm^-2, N4: 180 kg·hm^-2, and N5: 225 kg·hm^-2. The responses of soil microbial biomass C and N, soil soluble organic C and N to different application rates of N fertilizer were studied. 【Result】 Results from repeat measures ANOVA indicated that both sampling period and N rates had significant effects on SMBC, SMBN, SSOC and SSON contents (P＜0.01). The SMBC, SMBN, SSON were significantly affected by wheat varieties, but not the SSOC and SOC (P＞0.05). SMBC, SMBN, SSOC and SSON were higher in the N fertilized treatments than N0 treatment (P＜0.05), and were the highest in the N3 treatment and then decreased with the application rate of N, especially in the N5 treatment, the SSOC and SSON contents were lower than N0. The SMBC and SSON contents were significantly increased in N fertilizer treatments compared to SMBN and SSOC. In addition, while the responses of SMBC, SMBN, SSOC, and SSON to application rate of N were mostly quadratic or cubic equation. SMBC/SMBN was the lowest in the N3 treatment (the mean of two wheat varieties was 7.94 and 7.83, respectively) and the highest in the N5 treatment (the mean of two wheat varieties was 13.28 and 11.45, respectively). During the wheat growing period, the SMBC, SMBN, SSOC and SSON contents generally were the highest at flowering, followed by harvesting, jointing, tillering and seeding. In general, the impact of treatments (different application rates of N) on SMBC, SMBN, SSOC and SSON was more significant than seasonal fluctuation, and significant positive relationships were found between SSOC and SMBC and between SSON and SMBN (P＜0.01). There was no significant difference (P＞0.05) in aboveground biomass of wheat between Liaochun 10 and Liaochun 18 throughout most of the wheat growing season, but it was decreased slightly after a increase and tended to the peak in N3 treatment with the application rate of N; SOC and soil TN were the highest in N3 treatment and harvest period. 【Conclusion】 In this experiment, appropriate application rate of N fertilizer (N3, 135 kg·hm^-2) had a high ability to modulate the transformation of SMBC, SMBN, SSOC and SSON throughout the wheat growth period and sustainable soil productivity.

HORTICULTURE

Analysis of Basic Leucine Zipper Genes and Their Expression During Bud Dormancy in Apple (Malus×domestica)

SUN Ming-yue, ZHOU Jun, TAN Qiu-ping, FU Xi-ling, CHEN Xiu-de, LI Ling, GAO Dong-sheng

Scientia Agricultura Sinica. 2016, 49(7):  1325-1345.  doi:10.3864/j.issn.0578-1752.2016.07.010

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【Objective】In this study, putative bZIP transcription factor-encoding genes in the apple (Malus×domestica Borkh.) genome were identified, so as to provide a basis for studying the theoretical roles of bZIP genes in the bud dormancy, and to provide valuable information for bZIP genes in apple. 【Method】 The Hidden Markov Model profiles of the bZIP domains (PF00170 and PF07716) downloaded from Pfam were used to search the database using HMMER, ver. 3.0 with the default E-value. The obtained amino acid sequences were analyzed with the bioinformatics softwares, including Clustal Omega, MEGA6.0, MapInspect, DNAMAN 6.0 and MEME4.10.2. Furthermore, Microarray analysis and qRT-PCR results indicated that many MdbZIP genes may be involved in regulating bud dormancy in different cultivar of apples. 【Result】 In Total 120 MdbZIP genes were found in the apple genome. Phylogenetic analyses of the genes based on A. thaliana counterparts indicated that the transcription factors can be categorized into 10 different groups (Groups A–I and S), Chromosome mapping analysis showed that 109 MdbZIP genes were distributed unevenly on 17 chromosomes. Except for 11 genes that were not located on chromosomes, the largest number of MdbZIP genes were found on chromosomes 8 (thirteen genes), only 1 genes located on chromosome 1, some chromosomal regions had a relatively high density of MdbZIP genes. The results of gene structure analysis revealed that MdbZIP gene contained 0-23 exons, 23 bZIP genes were intronless and were found in Groups S (nine genes) and F (one gene), bZIP gene structure were highly conserved in apples. Conserved motif analysis showed that the conserved motifs 1, which specify the bZIP domain were observed in all apple bZIP proteins, motif 10 and 14 as the known domain were observed in Group D and G, respectively. Furthermore, microarray data indicated that many MdbZIP genes may be involved in regulating bud dormancy release. And qRT-PCR results indicated that ABA induce eight members of group A expression in high-chill and low-chill apples, but the largest number of differentially expressed genes was found in group D with cold temperatures. 【Conclusion】 These results suggested that MdbZIP gene family was highly and structurally conserved, and involved in abscisic acid (ABA) signaling and cold stresses thereby possibly involved into the regulation of bud dormancy in apples.

Biomarker Sieving for Fruit Storage Life of Satsuma Mandarin (Citrus unshiu Marc.)

SUN Li, DING Yu-duan, HE Yi-zhong, CHEN Ling-ling, CHENG Yun-jiang

Scientia Agricultura Sinica. 2016, 49(7):  1346-1359.  doi:10.3864/j.issn.0578-1752.2016.07.011

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【Objective】Experiments were carried out to study the physiological status of harvested Satsuma mandarin and to discover representative biomarker candidates that represent the status of citrus fruit. 【Method】Based on the data of microarray and primary metabolites of Satsuma mandarin during 0-50 DAH (days after harvest), approaches of hierarchical clustering, principal component analysis, partial least squares discriminant analysis and support vector machine model were integratively used to assess different physiological status and to discover representative biomarker candidates representing citrus fruit storage process. Additionally, the high reliability of transcriptome data was verified by qRT-PCR. 【Result】A total of 5 232 differentially expressed genes were identified. The post-harvest storage process of Satsuma mandarin could be classified into early stage, middle stage and terminal stage according to the differentially expressed genes. Functional categorization by Mapman showed that the biological process of Satsuma mandarin during storage was closely related to amino compound metabolism and cell wall metabolism. The statistical analysis of gene ontology revealed that nucleosome assembly genes and proteolysis genes were overrepresented. Combined with the verification of qRT-PCR, the expression trend of glutathione S-transferase had obviously stage characteristics, and therefore it can be used as a biomarker evaluating Satsuma mandarin post-harvest storage status. The same classification of physiological stages of Satsuma mandarin during storage was done according to the primary metabolites data. Further, four candidate metabolite biomarkers, including Glycine, 3-Amino-6-methoxypicolinic acid, 2-Butenedioic acid and Cyclohexanone,2-[(dimethylamino)methyl] were screened.【Conclusion】Five biomarkers, including glutathione S-transferase, glycine, 3-amino-6-methoxypicolinic acid, cyclohexanone,2-[(dimethylamino)methyl], 2-butenedioic acid were screened, which could be used to predict the storage life of harvested Satsuma mandarin.

STORAGE·FRESH-KEEPING·PROCESSING

Protein Phosphorylation on the Function of Myofibrillar Proteins in Mutton Muscle

CHEN Li-juan, LI Xin, LI Zheng, LI Pei-di, LI Zhong-wen, ZHANG De-quan

Scientia Agricultura Sinica. 2016, 49(7):  1360-1370.  doi:10.3864/j.issn.0578-1752.2016.07.012

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【Objective】The aim of this study was to analyze the effect of the phosphorylation level which was improved by activating the activity of protein kinase on myofibrillar proteins’ degradation and contraction.【Method】Improve the activity of protein kinases by adding protein kinase A activator Forskolin and protein kinase C activator PMA, followed by the increasing of the phosphorylation level of myofirillar proteins in mutton muscle. By comparing the myofibrillar fragmentation index (MFI), the degree of myofibrillar degradation, the sarcomere length, and other indicators between activator groups and control group to determine the effect of protein phosphorylation levels on lamb muscle contraction and degradation. 【Result】The activity of protein kinases in the PMA group (PKC activated group) were higher than the control group after 1 hour, 2 hour, and 4 hour incubation (P＜0.05). The activity of protein kinases in the Forskolin group (PKA activated group) were higher than in the control group after 1 hour and 4 hour incubation (P＜0.05). The highest activity of the protein kinases appeared after 1 hour incubation. PMA and Forskolin can improve the phosphorylation level of titin, myosin binding protein C, tropomyosin, myosin light chain 2, and a protein of 25 kDa by activating the protein kinases, but the phosphorylation level of myosin heavy chain and actin had no changes. The degradation degree of the PMA group and the Forskolin group were lower than the control group, and the sarcomere length of the PMA group and the Forskolin group was shorter than the control group. 【Conclusion】 On the one hand, the improvement of the myofirillar proteins phosphorylation level can reduce the degree of myofibrillar degradation, while on the other hand, the improvement of the myofirillar proteins phosphorylation level may enhance the force of muscle contraction through the effect on myosin light chain 2, may accelerate the contraction of filament through the effect on adjacent tropomyosin, and finally influence the rigor mortis and tenderization.

Fungal Diversity of Huaidao No. 5 Rice and the Dominant Culturable Fungal Strains During Storage

DU Li-hui, HE Xiao-ying, LIU Ling-ping, YUAN Jian, JU Xing-rong

Scientia Agricultura Sinica. 2016, 49(7):  1371-1381.  doi:10.3864/j.issn.0578-1752.2016.07.013

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【Objective】The objective of this study is to determine the fungal genus in 2013 Huaidao No. 5 rice by traditional morphological observation and molecular biology method, as well as to investigate the dominant fungal strains on Huaidao No.5 rice under different storage conditions. Results of this study could provide a scientific basis for the fungal genus identification and control of fungi growth in Huaidao No. 5 rice. 【Method】The culturable fungal strains were obtained by using both Rose Bengal and potato dextrose agar (PDA) media, following the instruction of GB 4789.15-2010. The Huaidao No. 5 rice collected from 4 different regions were mixed and then the moisture content were adjusted to 14.5% and 18.5% respectively. The samples were stored in the artificial climate equipment under 25℃. The culturable dominant fungi were determined by traditional culturing of the rice samples monthly removed from the simulated storage conditions. The fungal genus were identified by morphological observation of colonies, hyphae and spores by referencing to the . At the same time, the internal transcribed spacer (ITS) and 28S rDNA were amplified by PCR, and then ligated into T vector and sequenced. The fungal species were identified by constructing phylogenetic trees.【Result】In terms of fungal diversity in Huaidao No. 5 rice: 33 strains of fungi were isolated from four regions of the 2013 Huaidao No. 5 rice, mainly for Penicillium, Aspergillus and Fusarium, including 16 strains of Penicillium and 15 strains of Aspergillus, accounting for the separated strains of 48.5% and 45.5%, respectively, indicating that Penicillium and Aspergillus are the dominant fungi genus in 2013 Huaidao No. 5 rice. There are only 2 strains of Fusarium, which accounted for only 6% of the isolates. Simulation storage experiment results showed that Penicillium aurantiogriseum appeared to be the dominant strain from 6 month in moisture content of 14.5% for Huaidao No. 5 rice under 25℃ storage, and this strain persisted to be dominant in 7 and 8 months storage. While for the Huaidao No. 5 rice with a moisture content of 18.5%, a yeast strain appeared to be the dominant from 7 month and this strain persisted to be dominant at 8 month under 25℃ storage.【Conclusion】 33 strains of fungi were separated and identified from 2013 Huaidao No. 5 rice, including 16 strains of Penicillium and 15 strains of Aspergillus, respectively, indicating that Penicillium and Aspergillus are the dominant fungi genus in 2013 Huaidao No. 5 rice. And under different simulated storage conditions, different cultural fungal strains dominated the Huaidao No. 5 rice with different moisture content.

ANIMAL SCIENCE·VETERINARY SCIENCERE·SOURCE INSECT

Comparative Analysis of Growth Performance, Behavior, and Salivary Cortisol Hormone of Piglets Housed in Two Rearing Environments

ZHU Hong-long, YANG Jie, LI Jian, PAN Xiao-qing, QIN Feng, ZHOU Zhong-kai, FENG Guo-xing, GU Hong-ru

Scientia Agricultura Sinica. 2016, 49(7):  1382-1390.  doi:10.3864/j.issn.0578-1752.2016.07.014

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【Objective】The aim of this study is to make a comparative analysis of growth performance, behavior, and cortisol hormone of weaned piglets housed in two rearing environments.【Method】A total of 88 Suzhong piglets (half of castrated male and female) weaned at 35 d of age were randomly assigned into either the slatted-floor house (SFH) treatment or the deep-litter house (DLH) treatment. Each treatment had 4 pens (4 replicates), and according to the similar rearing density (0.67 m²/animal), each replicate for SFH and DLH had 6 and 16 piglets respectively. The experiment included 14 days of an adaption period and 21 days of an experimental period. Piglets had always access to feed and water. Feed intake was recorded during the experiment period, and piglets were weighed individually at the age of 49 and 70 days to get the growth performance according to replicate. At the age of 68 days, video was recorded for 24 h, and the three piglets from each replicate were selected for observation of piglets’ behavior during daylight from 07:00 to 17:00. Salivary samples of the three piglets from each replicate were collected from 09:00 to 10:00 for the cortisol analysis at the age of 69 days.【Result】Final weight, average daily feed intake, average daily gain, and feed/gain ratio of piglets from DLH did not differ from those of piglets from SFH (P＞0.05). Piglets from DLH spent significantly more time in standing and sitting (P＜0.05), and less time lying (P＜0.05) compared to piglets from SFH. The activities of piglets mainly occurred during 08:00-10:00 and 12:00-16:00. DLH piglets spent more time at locomotion and explorative behavior (particularly rooting deep bedding) (P＜0.05), and less time manipulating pen and aggressive behavior (P＜0.05), compared to SFH piglets. DLH increased the time spent drinking (P＜0.05), but had no effect on the time spent eliminating (P＞0.05). DLH decreased the total time spent feeding (P＜0.05), but DLH piglets exhibited higher average duration of feeding events due to their low number of feeding events. DLH increased the concentration of cortisol in saliva of weaned piglets (P＜0.05).【Conclusion】DLH could increase time spent in locomotion, explorative behavior, and average duration of feeding events and decrease time spent in aggressive behavior in weaned piglets. These data showed DLH could improve animal welfare in the piglet production.

Association of the RIPK2 Gene Genetic Variation with Ujumqin Sheep Growth Traits

MA Xiao-meng, XUAN Jun-li,WANG Hui-hua,YUAN Ze-hu, WU Ming-ming, ZHU Cai-ye, LIU Rui-zao, WEI Cai-hong, ZHAO Fu-ping, DU Li-xin, ZHANG Li

Scientia Agricultura Sinica. 2016, 49(7):  1391-1407.  doi:10.3864/j.issn.0578-1752.2016.07.015

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【Objective】Based on the results of the previous genome-wide association on the sheep meat traits, we studied the possibility of the RIPK2 gene to be a novel candidate gene for the sheep growth traits, in order to confirm the GWAS results, as well as to find new molecular markers related to the sheep growth traits.【Method】In the 343 Ujumqin sheep experiment group, 30 DNA samples with a fixed concentration were selected to construct the DNA pool, primers were designed to extend exons and 1 000 bp of upstream and downstream regulate regions. PCR products were tested and the objective strap were sequenced. The sequence peak maps were analyzed by DNAMAN and Chromas2 softwares, Sequenom Mass-array technology was used to genotype the detected SNP including the SNP obtained by previous GWAS. Haploview was used to construct haplotypes and analyze linkage disequilibrium of the polymorphic loci. Association analysis between the SNPs in RIPK2 gene and the growth traits were performed using SPSS 22.0 software. 【Result】 Four SNPs were detected. In exon 4 was a A5536G synonymous mutation rs01, there were three genotypes on this site and the frequencies of AA, AG and GG were 0.42,0.45 and 0.13 individually, the dominant allele was A with a frequency of 0.65. In exon 7 was a T8952C missen variation rs02 with three genotypes. The frequencies of TT, TC and CC genotypes were 0.82,0.16 and 0.02. T was the dominant allele and its frequency was 0.9. Rs05 was a T2836C mutation in exon 10 with only two genotypes TT and CC. The frequency of genotype TT was 0.25 as well as CC was 0.75. In the upstream regions a G5181C variation rs30 was detected with three genotypes GG, GC and CC. The frequency of heterozygous genotype GC was 0.50 and the other two genotypes CC, GG were 0.18 and 0.32 respectively. G was the dominant allele with its frequency was 0.58.The variation T4456C detected by GWAS was rs34 in this study and there were three genotypes. The frequency of TT genotype was 0.49, the frequency of TC was 0.37 and the frequency of CC was 0.14.T was the dominant allele with a frequency of 0.68. The χ² test indicated all the SNPs fit with Hardy-Weinberg equilibrium except rs34. Rs02 was in low polymorphic status (PIC＜0.25) and the other three SNPs were in moderate polymorphic status (0.25＜PIC＜0.5). Linkage disequilibrium analysis suggested that, rs01 and rs02 were strongly linked, the two sites constructed three haplotypes in the experiment population, AT was the dominant haplotype and the frequency was 0.645. Combining the genotypes of the two sites, we found 6 genotype combinations. AATT was the superior genotype and its frequency was 0.425. Single SNP correlation analysis revealed that: on rs01 site, individuals with genotype AA/AG genotype were significantly higher than those with GG genotype on four months body weight, chest girth and six months body height, chest girth (P＜0.05). On rs02 site, individuals with CC genotype were significantly higher on the four months weight, body height, chest girth and six months chest width (P＜0.05) than the individuals with TT and TC genotypes as well as individuals with CC genotype were extremely significantly different from individuals with TT and TC on the four months body length, six months body height and body length (P＜0.01). Individuals with GG genotype on rs30 site showed a significant difference with the individuals of the other two genotypes on the four months weight and chest girth (P＜0.05) and individuals with each genotype had a significant difference on the six months chest girth (P＜0.05). Individuals with each genotype of rs34 had significant difference on the four months weight (P＜0.05). Meanwhile, the combined genotype of rs01-rs02 correlation analysis revealed that: individuals with GGCC combined genotype was significantly different from individuals with the other five genotypes on the four months body weight, body height, body length, chest girth and six months body height, body length and chest width with (P＜0.05).【Conclusion】The results showed that the different SNPs and combined genotypes in RIPK2 gene had effect on Ujumqin sheep different growth traits with the SNPs being potentially valuable as genetic markers for economic traits in Ujumqin sheep.

Establishment and Application of a RT-PCR Assay for Detection of Newly Emerged Porcine Deltacoronavirus

ZHANG Fan-fan, SONG De-ping, ZHOU Xin-rong, HUANG Dong-yan, LI An-qi, PENG Qi, CHEN Yan-jun, WU Qiong, HE Hou-jun, TANG Yu-xin

Scientia Agricultura Sinica. 2016, 49(7):  1408-1416.  doi:10.3864/j.issn.0578-1752.2016.07.016

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【Objective】Porcine Deltacoronavirus (PDCoV) is a newly emerged coronavirus, which has caused diarrhea in pigs, especially in newborn piglets leading to high morbidity and mortality, such that it has become one of the most important causes of death in newborn piglets. In this study, we attempted to establish a reverse transcription-polymerase chain reaction (RT-PCR) assay for detection of newly emerged PDCoV in diarrheal pigs, and investigate the infection situation of PDCoV in diarrheal samples of pigs.【Method】Based on the result of multialignment analysis on the published PDCoV genome sequences on GenBank database, a pair of primers specific to the conserved nucleocapsid (N) gene of PDCoV was designed by using the online software Primer 3.0, and then a RT-PCR for detection of PDCoV was established by utilizing the designed primers. Diarrheal samples were tested by the established RT-PCR assay, and several positive PCR products were selected for cloning and sequencing. To analyze the phylogenetic relationships between PDCoV and porcine epidemic diarrhea virus (PEDV) as well as transmissible gastroenteritis virus (TGEV), a phylogenetic tree was constructed based on the partial N gene sequences of PDCoV obtained in this study and 18 reference PDCoV sequences from other countries/areas, and corresponding N gene sequences of representative PEDV and TGEV strains. To determine the specificity of the RT-PCR assay established, RNAs of PEDV, TGEV, porcine kobuvirus (PKoV), porcine astrovirus (PAstV), porcine reproductive and respiratory syndrome virus (PRRSV) and classical swine fever virus (CSFV) were tested. To evaluate the detection limit of the assay, a recombinant plasmid containing the N gene fragment was constructed and served as templates with 10-fold serial dilution starting from 1.0×10⁶ copies/μL to 1.0×10¹ copies/μL. Clinic diarrheal samples of pigs (n=249) from sows and piglets collected in Jiangxi Province from 2012 to 2015 were tested for the presence of PDCoV by the established RT-PCR, and some PDCoV positive amplicons were cloned and then sequenced for the specificity confirmation of the assay.【Result】A single expected DNA fragment band of 329 bp in size was amplified by using the designed primers. Sequences of the amplicons selected were subjected to a BLAST search against the GenBank database, and the results indicated that the sequences hit PDCoV with a nucleotide identity as high as 99.1%, demonstrating that the amplified targets were PDCoV. A phylogenetic tree was generated based on eight partial N gene of PDCoV obtained in this study, 18 reference PDCoVs from other countries/areas, and two representative PEDV and two selected TGEV sequences with corresponding fragments. All of the PDCoV strains were classified into a cluster, distinct from PEDV and TGEV strains. The eight PDCoV sequences obtained in this study showed that the highest similarity with a Korea PDCoV strain KNU14-04, but less similarity to two Hong Kong strains HKU 15-44 and HKU 15-155. No cross amplification for PEDV, TGEV, PKoV, PAstV, PRRSV and CSFV was observed, indicating the established RT-PCR assay was highly specific. The detection limit of 1.0×103 copies/µL of the assay was determined based on the templates of the recombinant plasmid with 10-fold serial dilution (from 1.0×10⁶ to 1.0×10¹ copies/µL), indicating the assay was sensitive. A total of 249 diarrheal fecal/intestinal samples of sows and piglets collected in Jiangxi Province, China from 2012 to 2015 were examined by the established assay, and the positive rate of PDCoV was 31.3% (78/249), and the samples from sows showed somewhat lower positive rate (27.8%) when compared with that from piglets (31.9%). PDCoV could be detected in samples as early as 2012.【Conclusion】In this study, a RT-PCR assay for detection of newly emerged PDCoV in swine was established and evaluated. The results from this study indicated that PDCoV is a frequently detected virus in diarrheal pigs in Jiangxi, China. The RT-PCR assay established in this study is valuable in terms of clinic diagnosis and epidemiological investigation of PDCoV.

RESEARCH NOTES

Cloning, Sequence and Expression Analysis of Phosphoenolpyruvate Carboxykinase Genes (PEPCKs) in Apple

LI Hui-feng, RAN Kun, CHENG Lai-liang, WANG Hai-bo, HE Ping, CHANG Yuan-sheng, LI Lin-guang

Scientia Agricultura Sinica. 2016, 49(7):  1417-1428.  doi:10.3864/j.issn.0578-1752.2016.07.017

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【Objective】The aim of this study is to characterize an ubiquitous enzyme of phosphoenolpyruvate carboxykinase (PEPCK) involved in many pathways of plant metabolism. The transcriptional level of MdPCKs in different tissues and under various stress treatments were determined to predict the related function of MdPCKs gene. 【Method】 The full-length cDNA sequences of MdPCK1 and MdPCK2 were isolatedby homologous alignment and RT-PCR confirmation, the obtained cDNA sequences and the deduced amino acid sequences were analyzed with bioinformatics methods; the promoters of MdPCK1 and MdPCK2 were cloned and the cis-acting regulatory elements were analyzed through PlantCARE database; the expression levels of MdPCK1 and MdPCK2 were detected in different tissues and under SA, JA, ABA, PEG, high temperature and low temperature treatments using qRT-PCR.【Result】The sequencing results showed that two cDNAs (designated as MdPCK1 and MdPCK2; GenBank Accession No. KT454964 and KT454965) contained an open reading frame (ORF) of 2 001 bp and 2 028 bp, and their ORFs encoded a protein with 666 and 675 amino acids, respectively. The results of gene structure analysis revealed that both of the MdPCK1 and MdPCK2 contained 12 exons and PEPCK gene structures were highly conserved in plant. Amino acid sequences and structure analysis indicated that both of the MdPCK1 and MdPCK2 contained conserved PEPCK ATP domain. The results of promoter analysis showed that there were multiple putative cis-acting elements involved in light responsiveness, circadian control, MeJA-responsiveness, salicylic acid responsiveness, abscisic acid responsiveness, defense and stress responsiveness, low- temperature responsiveness and heat stress responsiveness. qRT-PCR results showed that MdPCKs were constitutively expressed in all examined tissues. MdPCK1 and MdPCK2 had similar expression patterns during fruit development of ‘Taishan Gala’ apple. Under various stress conditions, MdPCK1 expression was induced by 100 μmol·L^-1 ABA and 100 g·L^-1 PEG, and MdPCK2 expression was induced by 150 μmol·L^-1 SA, 100 μmol·L^-1 ABA, 100 g·L^-1 PEG and low temperature.【Conclusion】Taken together, the above results indicated that MdPCK1 and MdPCK2 belonged to plant PEPCK family, their expressions could be induced by abiotic stresses.