Table of Content

16 June 2017, Volume 50 Issue 12

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CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS

Expression and Functional Analysis of Acireductone Dioxygenase Gene in Rice

XIONG Wei, YANG Bo, LIU WeiYin, WANG Quan, KONG XiaoCong, JIN YaJun, LIANG ShanShan, LUAN WeiJiang, ZHANG SiJu

Scientia Agricultura Sinica. 2017, 50(12):  2199-2208.  doi:10.3864/j.issn.0578-1752.2017.12.001

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【Objective】 The tolerance to biotic/abiotic stress is highly associated with grain yield, which is a very important goal in crop breeding. The objective of this study was to analyze the expression pattern of OsARD1, to investigate its function to abiotic stress, and to provide a theoretical basis for rice breeding. 【Method】 To analyze the expression of OsARD1 in different tissues, total RNAs were isolated from different organs. To analyze the expression pattern of OsARD1, RNAs were isolated from wild type seedlings at 14-day-old under different abiotic stress, including PEG, high salinity and mechanical damage. RT-PCR method was used to analyze the expression of OsARD1 in different organs and under abiotic stress. An overexpressing vector of OsARD1 was constructed and OsARD1 overexpression transgenic plants were produced following the Agrobacterium-mediated transformation procedure. Transgenic plants with empty vector were also generated as a control. For drought stress, T1 generation transgenic plants and wild type at 12-day-old were subjected to water deficit treatment and then cultured in nutrition solution for 3 days to recover. For PEG osmotic stress experiment, wild type and OsARD1-overexpressing (OsARD1-OV) seeds were planted in agar medium containing 5% PEG6000 whose osmotic potential was -0.05 MPa. For control group, no PEG was added into agar medium. The phenotypes of wild type and OsARD1-OV plants were observed and recorded. 【Result】The tissue-specific expression analysis showed that OsARD1 expressed highly in root and mature organs, especially in senescent organs. OsARD1 gene transcript levels were strongly induced by PEG6000, high salinity and mechanical damage. Six independent OsARD1-OV lines were generated by Agrobacterium-mediated transformation method. RT-PCR expression analysis showed that the expression level of OsARD1 was significantly increased in OsARD1-OV lines of T0 and T1 generation plant compared with that in wild type, suggesting that overexpression vector worked and inherited stably. The experiments of water deficit of OsARD1-OV of T1 generation and wild type plants were further carried out. The results showed that the wild type plants exhibited severe dehydration symptoms with rolled and withered leaves after five hours of water deficit treatment, while OsARD1-OV plants were growing normally. After 8 hours of water deficit treatment and recovery culture for 3 days, there were only 10% wild type plants survived, while 80% transgenic plants survived. In PEG6000 osmotic stress experiment, it was found that the shoot and root length of wild type and OsARD1-OV plants had no significant difference with that in control group. However, the growth of WT roots was strongly inhibited in 5% PEG6000 treatment group compared with OsARD1-OV lines, and the root length of wide type was shorter than that of OsARD1-OV lines, indicating that the overexpression of OsARD1 improved drought tolerance in rice.【Conclusion】OsARD1 displayed higher expression in roots and mature tissues of rice, and was strongly induced by PEG, high salinity and mechanical damage. Overexpression of OsARD1 significantly improved the tolerance to water deficit and osmotic stress in rice, suggesting that OsARD1 plays a key role in drought tolerance.

Transcription Abundances of Two Phytochrome C in Response to Different Light Treatments in Zea mays

NIU Xiang, GUO Lin, YANG ZongJu, SUN Lei, LI HongDan, YOU GuangXia, XU Hong, MENG FanHua, SHE YueHui, YANGJianPing

Scientia Agricultura Sinica. 2017, 50(12):  2209-2219.  doi:10.3864/j.issn.0578-1752.2017.12.002

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【Objective】To study the functions of phytochrome C genes in seedling de-etiolation and flowering regulation in maize (Zea mays L.), two phytochrome C genes of maize (ZmPHYC1 and ZmPHYC2) were selected from the NCBI database and analyzed by bioinformatic methods. The transcription abundances of two ZmPHYC genes was analyzed in different tissues and in response to light qualities, transitions from the dark to different light conditions, photoperiod treatment (long day and short day) by quantitative RT-PCR (qRT-PCR).【Method】B73 inbred line was used in this study, the full length ORFs of two ZmPHYC genes were cloned by RT-PCR. The proper clones were sequenced and analyzed by bioinformatics software. The transcription abundances of two ZmPHYC genes in different tissues and in response to light treatments was detected using qRT-PCR.【Result】Both the full length ORFs of ZmPHYC1 and ZmPHYC2 contained 3408 nucleotides and encoded two polypeptides with 1135 amino acid motifs, and their molecular weight was 126.14 kD and 126.07 kD, respectively. Two ZmphyC proteins were able to be further divided into six domains: Per (period circadian protein)-Arnt (Ah receptor nuclear translocator protein)-Sim (single-minded protein) (PAS), cGMP-stimulated phosphodiesterase (GAF), phytochrome (PHY), PAS-related domain (PRD) containing two PAS, His Kinase A domain (HisKA), Histidine kinase-like ATPases (HATPase_c), while ZmphyC2 lacked a PAS in PRD domain. Phylogenetic analysis indicated that the two ZmphyC proteins belonged to the same branch with phyC proteins from other graminaceous species, especially with the phyC proteins from sugarcane and sorghum. qRT-PCR assays showed that both ZmPHYC1 and ZmPHYC2 belonged to tissue-specific genes and highly expressed in roots and leaves. The transcription abundances of the both genes were very high under blue and white light conditions. Both ZmPHYC1 and ZmPHYC2 displayed similar expression patterns during transitions from the dark to different light conditions. The transcription abundances of the both genes went dramatically up at 0.5 h after transitions from the dark to far-red, red, blue, or white light. And then they quickly dropped and waved below their own levels in the dark. Both ZmPHYC1 and ZmPHYC2 were also able to respond to long-day or short-day treatments. During long-day treatment, they likely had one peak in either light or dark period. During short-day treatment, they showed different expression patterns, the peak of ZmPHYC1 happened at 6 h after conversion to dark period, while ZmPHYC2 occurred at 2 h after conversion to light period.【Conclusion】 ZmphyC1 protein kept six domains, while ZmphyC2 lacks a PAS in PRD domain. The transcription abundances of the both PHYC genes were tissue-specific in maize. Similar expression patterns of ZmPHYC1 and ZmPHYC2 genes in response to different light treatments suggest that they both might keep redundant functions. Since the transcription abundances of ZmphyC1 were higher than these of ZmphyC2, ZmphyC1 might have more important role in seedling responding to light than ZmphyC2, which may be due to the existence of different functions in maize. Both ZmPHYC1 and ZmPHYC2 respond to light and photoperiod treatments, suggesting that they are involved in seedling de-etiolation and flowering time control in maize. Thus their roles in crop improvement are worthy of more exploration in the future.

The Expression Patterns and Function Analysis of GhFLP5, a Gene Related to Flowering in Upland Cotton (Gossypium hirsutum L.)

WANG CongCong, ZHANG XiaoHong, WANG XiaoYan, ZHANG Pan, FAN ShuLi, PANG ChaoYou, MA QiFeng, WEI HengLing, WANG HanTao, SU JunJi, YU ShuXun

Scientia Agricultura Sinica. 2017, 50(12):  2220-2231.  doi:10.3864/j.issn.0578-1752.2017.12.003

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【Objective】To provide more information for breeding, the gene Flowering Promoting Factor1-like Protein 5 (GhFLP5), a member of the flowering promoting factor family in Gossypium hirsutum L., was cloned, and then its expression patterns were studied to characterize its functions in flowering process. 【Method】According to the sequences on NCBI , specific primers were designed by Oligo7 and GhFLP5 was cloned from cDNA of cultivar CCRI50. The protein properties were predicted via ExPASy. FPFs of other species were retrieved from NCBI. ClustalX2 was used for multiple sequence alignment and phylogenetic tree was constructed by MEGA6. The samples of different stages and different tissues of early maturity variety CCRI50 and later maturity variety Lu28 were used to study the spatial and temporal expression profiles of GhFLP5. A 1 500 bp fragment upstream the start codon was taken from the genomic database, then the cis-acting elements were analysed with PlantCARE. Based on the predictions, several phytohormones were selected to explore the responses of GhFLP5. pBI121-GhFLP5, a recombinant expression plasmid was constructed and transformed into Arabidopsis. The homozygous overexpression lines were observed and expression profiles were performed with quantitative real-time PCR (qRT-PCR). 【Result】With a 300 bp coding frame, GhFLP5 encoded a 11.4 kD protein. There were three pretty conserved domains, which reveals that GhFLP5 has a close relationship with those of Glycine max, Medicago truncatula and Polulus trichocarpa. Spatial expression patterns showed that it expressed predominantly in leaves. And GhFLP5 was transcribed at a higher level in early maturity variety CCRI50 than in later maturity variety Lu28. Temporal expression patterns showed that it hit a peak at three-true-leaf stage in CCRI50 but at four-true-leaf stage in Lu28. There were mainly two kinds of cis-acting elements in promoter region: one was light-response elements and circadian elements, and the other was stress-response elements. According to the elements' distributions and functions, salicylic acid (SA), abscisic acid (ABA) and jasmonic acid (JA) were selected to treat cotton seedlings. As a result, GhFLP5 was activated by SA and ABA, while it was suppressed by JA. The overexpression plants bolted about 9 days and flowered about 7 days earlier than the wild type. Meanwhile, the rosette leaves decreased, and all the differences were extremely significant. Quantitative analysis showed that the genes promoting flowering such as LEAFY (AtLFY), SUPPRESSOR OF OVEREXPRESSION OF CONSTANS (AtSOC1), FLOWERING LOCUS T (AtFT), APETALA1 (AtAP1)and FRUITFULL (AtFUL) were up-regulated in 35S::GhFLP5 lines, while the gene AtFLC, delaying flowering, was down-regulated. Moreover, the auxin-responsive genes SMALL AUXIN UPREGULATED 20 (AtSAUR20) and SMALL AUXIN UPREGULATED 22 (AtSAUR22) were induced in transgenic Arabidopsis lines. GIBBERELLIN 20-OXIDASE 1 (GA20OX1), a gene involved in gibberellin (GA) biosynthesis, was also up-regulated more than two folds. 【Conclusion】The Arabidopsis thaliana lines over-expressing GhFLP5 performed an obvious early-flowering phenotype. Moreover, the gene expression profiles indicated that GhFLP5 may play dual roles in the transition to flowering via both GA and IAA signaling pathways.

MAIZE NUTRITION PHYSIOLOGY AND FERTILIZATION

Guidance: High Nitrogen Use Efficiency of High-Yielding Maize

LIU Peng, DONG ShuTing, LI ShaoKun, ZHANG JiWang

Scientia Agricultura Sinica. 2017, 50(12):  2232-2237.  doi:10.3864/j.issn.0578-1752.2017.12.004

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Characteristics of Dry Matter and Nitrogen Accumulation for High-Yielding Maize Production Under Irrigated Conditions of Shaanxi

ZHANG RenHe, WANG BoXin, YANG YongHong, YANG XiaoJun, MA XiangFeng, HANG XingHua, HAO YinChuan, XUE JiQuan

Scientia Agricultura Sinica. 2017, 50(12):  2238-2246.  doi:10.3864/j.issn.0578-1752.2017.12.005

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【Objective】The objective of this paper is to study the dry matter and nitrogen accumulation in high-yielding spring maize under irrigated conditions of Shaanxi in order to realize high and stable yield in this area. 【Method】A field experiment was conducted by different agronomic managements with the high-yielding variety shandan609 as materials from 2013 to 2015. High yielding cultivations were practiced, and then the yield and yield component, LAI, SPAD, characteristics of dry matter and nitrogen accumulation were analyzed based on the maize high-yielding cultivation. 【Result】The average yields under farmers’ practice, higher yielding cultivation, super high yielding cultivation were 11.1, 13.1 and 16.1 t·hm^-2, respectively, and 18.0% and 45.1% higher than those of control. Compared with the control, the higher yielding and super high-yielding cultivation had lower kernels per ear and thousand-kernel weights, but produced more ear number per hectare. More ears were the key to achieve maize high yield potential. The harvest indexes of higher yielding and super high-yielding cultivation were higher than that of farmers’ practice. Similarly, compared with the control, the higher yielding and super high-yielding cultivation showed more dry matter and nitrogen accumulation from silking to maturity and at maturity. In the super high-yielding cultivation, 41.8% greater dry matter production and 24.5% more nitrogen uptake after silking contributed 20.1% more to grain yield and 61.6% to grain nitrogen. Compared with the control, the higher yielding and super high-yielding cultivation also significantly increased LAI and SPAD values after silking. Grain yield was highly correlated with post-silking dry matter accumulation (r=0.988), and post-silking nitrogen accumulation (r=0.927). 【Conclusion】The results indicate that higher grain yield can be achieved by using integrated and optimized cultivation techniques under irrigated conditions of Shaanxi. The super high-yielding cultivation of spring maize has stronger photosynthetic potential, more dry matter and nitrogen accumulation (especially post-silking) and post-silking dry matter and nitrogen accumulation contributing to grain yield, thus providing a basis for production of super high-yield maize. The present study highlighted the benefits of integrating nutrient and agronomic management with matching the supply and demand of nitrogen to achieve maize high yield under irrigated conditions of Shaanxi.

Effects of Close Planting and Nitrogen Application Rates on Grain Yield and Nitrogen Utilization Efficiency of Different Density-Tolerance Maize Hybrids

LI GuangHao, LIU Juan, DONG ShuTing, LIU Peng, ZHANG JiWang, ZHAO Bin, SHI DeYang

Scientia Agricultura Sinica. 2017, 50(12):  2247-2258.  doi:10.3864/j.issn.0578-1752.2017.12.006

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【Objective】The objective of this experiment is to study the effects of close planting and nitrogen application rates on grain yield and nitrogen utilization efficiency of different density-tolerance maize hybrids. 【Method】Two summer maize cultivars, density-resistant hybrid (ZD958) and non-density resistant hybrid (LD981), were used as experiment materials to study the effects of different planting densities ( 52 500, 82 500 plant/hm²) and nitrogen rates (0, 90, 180, 270, 360 kg N·hm^-2) on dry matter accumulation, nitrogen translocation efficiency, nitrogen use efficiency, yield and its components of different density-tolerance summer maize.【Result】 The 1000-grain weight and kernels per ear were significantly decreased with the increase of planting density at the same nitrogen application level, but the ear number, barrenness and lodging rate were significantly increased. The barrenness and lodging rate of non-density resistant hybrid were increased more significantly. The average 1000-grain weight and kernels per ear of ZD958 and LD981 were decreased by 6.24% and 6.77%, 7.52% and 18.09%, respectively, and barrenness and lodging rate of LD981 were as high as 17% and 27.6%, significantly higher than ZD958. The grain yield increased with increase of N application rate under high density condition, but the difference between N application rate at 270 kg·hm^-2 and 360 kg·hm^-2 was not significant. Under low density condition, the grain yield increased first and then decreased with increase of N application rate, and reached the maximum at N application rate of 270 kg·hm^-2. The dry matter accumulation per plant decreased with the increase of planting density, while the population dry matter accumulation increased. Both of them increased with increase of N application rate, and the dry matter contribution rate increased after anthesis. Under the same nitrogen level, the high density treatments significantly increased the total N accumulation, N translocation and its contribution rate to grain. With the increase of planting density, the average total N accumulation, N agronomic efficiency and nitrogen utilization efficiency of ZD958 and LD981 were increased by 15.94%, 39.01%, 26.22% and 1.96%, 5.79%, 14.92%, respectively. Under the same planting density, the increase of nitrogen rate could improve the total N accumulation and assimilating amount of nitrogen after anthesis, while the nitrogen agronomic efficiency, nitrogen utilization efficiency and nitrogen partial factor productivity were decreased. With increase of planting density, the N translocation rate and N translocation rate of nutrient organs increased significantly. Under high planting density condition, the N translocation efficiency and contribution rate increased with increase of N application rate, while it decreased under low planting density condition. 【Conclusion】Under this experimental field condition, increased density and nitrogen application rate could significantly improve the dry matter accumulation of ZD958 and LD981. The effect of density on grain yield was significant between the two summer maize cultivars. Under the conditions of high density, increasing the amount of N fertilizer, the yields of two cultivars were increased significantly, while barrenness and lodging rate of LD981 increased significantly, which was the main reason for limiting grain yield increasing. Increasing density could significantly improve the nitrogen utilization rate and N translocation of vegetative organs. N assimilating amount after anthesis increased with increasing density in ZD958, and decreased in LD981. Nitrogen use efficiency decreased with increasing nitrogen application, but which could increase plant N uptake and nitrogen assimilation after anthesis under high density. combination of density and nitrogen could improve the yield and nitrogen utilization rate together. As far as the grain yield and nitrogen efficiency are concerned, the most optimal plant density and nitrogen rate of ZD958 were 82 500 plants/hm² and 270 kg·hm^-2, and the most optimal plant density and nitrogen rate of LD981 were 52 500 plants/hm² and 180 kg·hm^-2.

Effect of Different Maize Varieties and Nitrogen Supply on Root Characteristics and Nitrogen Uptake and Utilization Efficiency

CHENG Yi, WANG HongZhang, LIU Peng, Dong ShuTing, ZHAO JiuRan, WANG RongHuan, ZHANG JiWang, ZHAO Bin, LI Geng, LIU YueE

Scientia Agricultura Sinica. 2017, 50(12):  2259-2269.  doi:10.3864/j.issn.0578-1752.2017.12.007

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【Objective】Through the study of the relationship of maize root characteristics and nitrogen uptake and utilization efficiency, shoot biomass and yield formation to prove the effect of root morphological characteristics and nitrogen uptake capacity of maize yield. And then provide theoretical basis for the high yield and efficiency of maize production.【Method】The experiments were conducted in 2014-2015 at the Technological Innovation Center of Maize in Huang-Huai-Hai Region (36°18' N, 117°12' E) and the State Key Laboratory of Crop Biology, located at Shandong Agricultural University in Taian. To explore the effect of different maize varieties and nitrogen supply on root characteristics and nitrogen uptake and utilization efficiency, using Jingke 968 (JK968), Zhengdan 958 (ZD958) and Xianyu 335 (XY335) as the experimental materials, setting two nitrogen levels, 1.5 g/plant (low nitrogen, LN) and 4.5 g/plant (high nitrogen, HN), sampling root and shoot of plant at tasseling stage and maturity stage for determination of the related indexes of root system (the root dry weight, root length, root surface area, root volume), dry matter and nitrogen accumulation and distribution in soil column tests.【Result】The results showed that grain yield and biomass per plant, each index of root, nitrogen accumulation amount per plant, nitrogen translocation rate, nitrogen harvest index and use efficiency of JK968 were all significantly higher (P＜0.05) than those of XY335 and ZD958. The biomass, grain yield, N accumulation amount per plant of JK968 were higher than those of XY335 and ZD958 by 15.2%, 17.7%, 9.0% and 31.6%, 44.1%, 31.4%, respectively, under LN level, 5.4%, 12.9%, 8.9% and 13.5%, 26.8%, 23.5%, respectively, under HN level. Compared with LN level, the biomass, grain yield, N accumulation amount per plant at the HN level of JK968, XY335 and ZD958 increased by 15.7%, 10.2%, 33.9% and 26.5%, 14.8%, 34.0% and 4.3%, 25.1%, 42.5%, respectively. The root dry weight, root length, root surface area, root volume of JK968 at tasseling stage are higher than those of XY335 and ZD958 by 41.8%, 9.0%, 47.1%, 24.0% and 63.2%, 41.6%, 60.4%, 105.1%, respectively, under LN level, 24.3%, 6.0%, 35.2%, 19.7% and 40.3%, 30.0%, 49.3%, 78.7%, respectively, under HN level. Compared with LN level, the root dry weight, root length, root surface area, root volume at the HN level of JK968, XY335 and ZD958 increased by 48.3%, 37.3%, 36.4%, 12.7% and 69.1%, 41.3%, 48.4%, 16.7% and 72.5%, 49.7%, 46.5%, 29.3%, respectively. The correlation analysis indicated that the amount of N-uptake showed a significant positive linear correlation with the root dry weight, root length, root surface area and root volume. The root index of different cultivars showed different responses to nitrogen, which the responsivity of each root index of JK968 to nitrogen was higher before tasseling but lower after tasseling than XY335 and ZD958. 【Conclusion】As for JK968, the root indexes of the whole growth period were significantly higher than those of XY335 and ZD958, it had a stronger nitrogen uptake ability and larger biomass, which were more distinct at the low nitrogen level. All these indicate that the larger root system of JK968 can ensure the nitrogen uptake to have higher nitrogen transportation efficiency, nitrogen contribution rate and nitrogen utilization efficiency, making for its material production, and finally obtaining a higher grain yield.

Effects of Different Cultivation Patterns on Root Characteristics, Yield Formation and Nitrogen Utilization of Summer Maize

REN Hao, CHENG Yi, LIU Peng, DONG ShuTing, ZHAO Jie, ZHANG JiWang, ZHAO Bin

Scientia Agricultura Sinica. 2017, 50(12):  2270-2281.  doi:10.3864/j.issn.0578-1752.2017.12.008

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【Objective】The objective of this study was to promote the growth of maize roots, enhance root absorption performance, and then reduce the amount of fertilizer, and then provide a theoretical basis for promoting the production of high yield and high efficiency by investigating the effects of different cultivation patterns on the root performance, grain yield formation and nitrogen uptake and utilization of summer maize, and exploring the relationship between root morphological characteristics and nitrogen uptake capacity of maize under different cultivation patterns. 【Method】Under two soil fertilities, with no nitrogen treatment as control (CK), three cultivation patterns including super high-yielding cultivation pattern (SH), high-yielding and high efficiency cultivation pattern (HH) and local farmer's practice(FP) in the long-term experiments by using ZD958 as testing varieties. The regulation of root characteristics on yield formation and nitrogen utilization in summer maize under different cultivation patterns were measured in fixed field experiment. 【Result】The yields of summer maize was significantly different among different cultivation patterns, compared with the HH, FP and CK patterns, the average yield of SH pattern in two years both in high soil fertility (HSF) and low soil fertility (LSF) were increased by 3.54%, 17.50%, 30.12% and 3.16%, 18.45%, 27.72% respectively. Multivariate analysis showed that both soil fertility and cultivation patterns had significant effects on summer maize yield, and comprehensive effect of double factors varied by years. The population biomass in high and low soil fertilities was SH > HH > FP > CK during the whole growing season. The total nitrogen accumulation in SH pattern was significantly higher than those in other patterns at the VT and R6 stages, and the N use efficiency and agronomic N use efficiency were higher than that of FP. HH pattern had the highest agronomic N use efficiency, nutrient use efficiency and nitrogen harvest index. Its nitrogen partial factor productivity was lower than FP, but still higher than the SH pattern. The results showed that summer maize root growth was greatly affected by different cultivation patterns. Root dry weight density, root length density and root surface area density were all expressed as SH > HH > FP > CK. From V12 to VT stage, increases in root length, root dry weight and root surface density of SH and HH patterns were higher than that of FP pattern, and decreases in that of FP pattern were highest from VT to R3 stage. The proportion of active absorption area of SH and HH patterns at VT stage was significantly higher than that in FP pattern. The root length density, root surface area density, root dry weight density were very significantly and positively correlated with yield and nitrogen use efficiency. 【Conclusion】HH pattern effectively promoted root development, delayed the senescence of root, improved the nitrogen use efficiency, achieved the high yield and high efficiency of summer maize.

Effect of Nitrogen Application on Carbon and Nitrogen Metabolism of Different Summer Maize Varieties

CHANG JianFeng, DONG PengFei, WANG XiuLing, LIU WeiLing, LI ChaoHai

Scientia Agricultura Sinica. 2017, 50(12):  2282-2293.  doi:10.3864/j.issn.0578-1752.2017.12.009

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【Objective】The study is designed to put forward the physiological indexes of high and stable yield in maize and explore the new management ways by comparing the differences of carbon and nitrogen metabolism of different maize varieties under different nitrogen levels and analyzing its coordination. 【Method】 The study was carried out to compare the differences of yield, the staying-green duration of leaf, nitrogen uptake and translocation, the ratio of C/N, PEP carboxylase and RUBP carboxylase activity by field and pot experiments in Wenxian and Zhengzhou from 2009 to 2011, in which 4 varieties of summer maize, including ZD958, ND108, XD20 and YD2002, were planted at 60 000 plants/hm² under four nitrogen levels of 0, 120, 240, 360 kg·hm^-2.【Result】(1) The grain yield of 4 maize varieties increased significantly with nitrogen level increasing. In pot experiments, the yield gap of XD20 between different nitrogen levels was the biggest, and that of ZD958 was the smallest. However, under the field condition, the difference of maize except XD20 between N240 and N360 was not significant. (2) The comparison of nitrogen uptake and translocation during grain-filling stage indicated that the nitrogen in ZD958 vegetative organs could be preferentially transferred to the leaves under low nitrogen condition which contributed to the longer staying-green duration of maize leaf. For ND108, both of nitrogen uptake and translocation were larger, thus its leaf can also stay green longer, but the effective utilization of nitrogen was lower. For XD20, due to lack of mechanism of priority supply to leaves and lower N uptake of under low nitrogen condition, the leaf showed early senescence. For YD2002, because of larger nitrogen transfer amount from vegetative organs to grain under four nitrogen levels and smaller nitrogen absorption in root, the leaf also showed early senescence. (3)During the grain filling stage, the C/N ratio of leaf in ZD958 and XD20 were between YD2002 and ND108 in the condition of normal fertilization. For the mature stage, the grain C/N ratio of ZD958 and XD20 were higher than the other two varieties. (4)At mid filling stage, ratio of PEPCase and RUBPCase of ZD958 was significantly higher than the other three cultivars, that of YD2002 was lower, and that of XD20 and ND108 increased significantly with the increase of nitrogen fertilizer. 【Conclusion】The excellent coordination of carbon and nitrogen metabolism of maize was reflected in the translocation process of carbohydrate and nitrogen. The maize that be able to coordinate the contradiction between grain and vegetative organ demand for carbohydrate and nitrogen, and with a higher PEPCase/RUBPCase, could reach a high and stable yield.

Effects of Nitrogen Application Times on Stem Traits and Lodging of Summer Maize (Zea mays L.) in the Huang-Huai-Hai Plain

BIAN DaHong, LIU MengXing, NIU HaiFeng, WEI ZhongBo, DU Xiong, CUI YanHong

Scientia Agricultura Sinica. 2017, 50(12):  2294-2304.  doi:10.3864/j.issn.0578-1752.2017.12.010

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【Objective】 Stem lodging is an important adversity affecting summer maize in the Huang-Huai-Hai China Plain under current husbandry practices, and may limit progress towards the achievement of higher yields via increased plant population density. For this problem, the influence of nitrogen application time on the stem development and lodging property of summer maize was studied under field conditions for two successive years. The aim of the experiment is to provide a scientific basis for the nitrogen management of summer maize under high-yielding conditions to improve the stem lodging-resistance capability in the Huang-Huai-Hai Plain.【Method】The experiment was conducted with three different morphological characteristics hybrids and six nitrogen application times from 2011 to 2012. The hybrids were Xianyu335 with higher plant height and lower ear height (hereafter refers to as a XY), Xundan20 with medium plant height and higher ear height (hereafter refers to as a XD) and Jingdan28 with medium plant height and lower ear height (hereafter refers to as a JD). The nitrogen application times included seed manure (N1), seedling fertilizer (N2), jointing fertilizer (N3), big-bell stage fertilizer (N4), tasseling stage fertilizer (N5), and no nitrogen treatment (N0) as the control.【Result】The results showed that nitrogen application time had significant impacts on the morphological, anatomical and mechanical characteristics of summer maize stem (p＜0.05). N1, N2 and N3 treatments promoted the stem growth which the ear height coefficient and center gravity of plant, diameter, stem wall thickness, cortex thickness, big vascular bundles number and small vascular bundles number, stalk lateral breaking strength and stalk rind penetration strength of the third internodes and bending strength of plant were significantly higher than N0. The ratio of the length to diameter of the third internodes and the field lodging rate significantly reduced in N1 and N2, while slightly increased in N3 as compared to N0. There were no significant differences in the ear height coefficient and internode length under N4 treatment, but the diameter, anatomical and mechanical characteristics of the third internode were significantly higher than N0, and the ratio of internode length to diameter was slightly lower. And the field lodging rate was also significantly lower than N0. Nitrogen application at the tasseling stage had no differences on stem growth of summer maize, and the field lodging rate significant reduction was induced by the reduction of ear height coefficient and center gravity of plant. Furthermore, it was found that the grain weight, kernel numbers per ear and yield of summer maize under all the nitrogen application treatments were significantly higher than N0. The yield increasing rate of N3 and N4 which had higher grain weight and kernel number per ear were the first, followed by N1 and N2 which had more kernel number per ear and lower grain weight, and then was N5 which had higher grain weight and significant lower kernel number per ear. 【Conclusion】 The reasonable nitrogen application time could promote the stem growth of summer maize, significantly reduced the ratio of the length to diameter of the basal internodes and improved the stem lodging-resistance capability. The effects of N1 and N2 were the best, but the yield increasing rate was a little lower due to lower grain weight. The stem lodging-resistance capability was lower in N3 which resulted from the higher ratio of the internode length to diameter, and should be avoided in the summer maize culture. The stem lodging-resistance capability, yield and yield components in N4 were the best. Therefore, combining with previous researches, split nitrogen application with small amount in N1 or N2 while with high ratio in N4 is beneficial to promote stem and ear of summer maize growth robustly, and then promote grain yield and stem lodging-resistance of plant.

PLANT PROTECTION

Recent Advances in Taxonomy of Plant Pathogenic Bacteria

FENG Jie

Scientia Agricultura Sinica. 2017, 50(12):  2305-2314.  doi:10.3864/j.issn.0578-1752.2017.12.011

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With the deepening of the research on plant pathogenic bacteria, taxonomy on plant pathogenic bacteria has been in a changing process. In addition to the traditional genera were divided into several genera, new genus has been also increasing to nearly 40 genera. Four genera were separated from Erwinia in gram-negative bacteria, such as Pectobacterium, Dickeya, Pantoea and Brenneria. Pectobacterium carotovorum and P. atrosepticum causing soft rot diseases of several crop plants, were originally classified as Erwinia carotovora subsp. carotovora and subsp. atroseptica, respectively. The genera of Ralstonia, Burkholderia and Acidovorax were separated from Pseudomonas. The naming of new species is also emerging one after another. Now, there are 8 species in Dickeya, the bacteria causing maize bacterial stalk rot and rice bacterial foot rot were reclassified as D. zeae. Some subspecies or pathovar have been upgraded to species. Acidovorax citrulli causing bacterial fruit blotch were originally classified as A. avenae subsp. citrulli. The Latin names of many pathogenic bacteria had experienced several changes. Xanthomonas axonopodis pv. malvacearum causing cotton angular leaf spot were originally classified as X. campestris pv. malvacearum or X. citri pv. malvacearum. Members of the gram-positive bacteria family are also growing. Domestic scholars exist outdated and nonstandard phenomenon in using Latin names. The purpose of this review is to present taxonomy and recent changes of plant pathogenic bacteria, and show the changing process of the old and new Latin names. The focus on plant pathogenic bacteria taxonomy will help to in line with international convention.

Effects of Transgenic Maize on Biodiversity of Arthropod Communities in the Fields

REN ZhenTao, SHEN WenJing, LIU Biao, XUE Kun

Scientia Agricultura Sinica. 2017, 50(12):  2315-2325.  doi:10.3864/j.issn.0578-1752.2017.12.012

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【Objective】 The objective of this study is to assess the effects of transgenic glyphosate-tolerant (EPSPS) and insect-resistant (Bt Cry1Ab) maize DBN9936 on biodiversity of arthropod communities, which will supply basic data for transgenic crop biosafety. 【Method】 In the growing period in 2015, the biodiversity and its structure of arthropod communities in the fields of transgenic maize and its counterpart lines were compared in the experimental base in Yitong Autonomous County for Man Nationality, Siping City, Jilin Province. In the isolated maize field, with the method of observation, traps and observing the longitudinal section of stems, the number of species and individuals in the maize plants, on the ground and in the stems were counted and the functional groups, biodiversity index, dominant concentration index, evenness index and the community similarity were analyzed and compared. 【Result】The arthropod communities in maize fields were composed of 13 orders and 44 families. The main pests included Rhopalosiphum maidis, Monolepta hieroglyphica and Osrtinia furnacalis; the main predatory enemies were lacewings, ladybirds and spiders. The number of target lepidoptera insects, including O. furnacalis and Helicoverpa armigera, from the transgenic maize DBN9936 in the whole growth period was significantly lower than those from the counterpart DBN318 and the conventional line Xianyu 335. The number of M. hieroglyphica from the transgenic maize was similar with the recipient line DBN318, while significantly higher than that from conventional line Xianyu 335, which showed the difference coming from variation between lines, rather than the genetically modification. The number of non-target arthropod in the fields had no significant difference between the other treatments. Sampling at the time of 2 and 4 weeks after spraying glyphosate, the number of specie and individual of arthropod communities in the fields had no significant difference between each two treatments. The number of boring insects and their damage were investigated during harvest, the number of holes, alive pests, the length of tunnel and the number of corns with damaged tip were significantly lower in DBN9936 than those in DBN318 and Xianyu 335, which showed that the transgenic maize DBN9936 had obviously higher resistance to O. furnacalis and H. armigera. Compared with the non-transgenic recipient line maize DBN318 and the regular line Xianyu 335, transgenic maize DBN9936 had the similar dynamic trend within the parameters of the number of species, biodiversity index, dominant concentration index and evenness index, and there was no significant difference. The similarity index of arthropod communities of DBN9936 with or without the herbicide to the recipient line DBN318 were gradually rising to a high level and maintained, which indicated that the difference of the arthropod communications from different treatments would be decrease in growing period.【Conclusion】In the study, the biodiversity dynamics of the arthropod communities of different treatments were similar. Combined climate recordings and field observation, the fluctuation of the dynamics from late July to mid August in 2015 could be regarded as the biodiversity decrease, especially the increase of dominant concentration index and the decrease of evenness index caused by the drought and the outbreak of M. hieroglyphica. These results suggested that the arthropod community structures in the fields of the transgenic maize DBN9936, the counterpart DBN318 and the conventional line Xianyu 335 were similar, and the effects of genetically modified maize on non-target arthropod community were not significant.

HORTICULTURE

Review of Melatonin in Horticultural Crops

GONG Biao, SHI QingHua

Scientia Agricultura Sinica. 2017, 50(12):  2326-2337.  doi:10.3864/j.issn.0578-1752.2017.12.013

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Melatonin is a kind of indoleamine compound that is widely existed in organism. In animal, melatonin acts as a health care product for human and plays a role in the regulation of circadian rhythm, improving immunity and anti-aging. Melatonin has been detected in a number of plant species up to now. And the biosynthesis pathway of melatonin includes L-tryptophane, tryptamine, 5-hydroxytryptamine and N-acetyl-5-hydroxytryptamine. The key enzymes involving melatonin biosynthesis have been detected in plants, including L-tryptophan decarboxylase (TrpDC), tryptophan hydroxylase (T5H), serotonin-N- acetyltransferase (SNAcT), 5-serotonin-N-acetyltransferase (AcSNMT) and hydroxyindole-O-methyltransferase (HIOMT). The roles of melatonin in the horticultural crops kingdom are not clear enough. In recent years, several studies showed that melatonin has roles in regulating the growth of plants, increasing yield, activating seed germination, regulating photoperiod, regulating rhizogenesis, delaying leaf senescence, influencing fruit ripening and storage. The antioxidant properties of melatonin would seem to explain, at least partially, its ability to fortify plants reactive oxygen species (ROS) scavenging that subjected to stresses, such as light, temperature, water, saline-alkali, heavy metal and oxidative stress. In addition, melatonin also involves some signaling transduction pathways including auxin (IAA), gibberellic acid (GA), abscisic acid (ABA), ethene (ETH), salicylic acid (SA), polyamine (PAs) and nitric oxide (NO), which form a complicated signaling network of growth, development and stress tolerance in horticultural crops. Recent data on five fields of “the biosynthesis of melatonin in plants, the melatonin of horticultural crops and influence factor for their melatonin content; roles of melatonin in growth and development of horticultural crops; roles of melatonin in stress response of horticultural crops, signal transduction network of melatonin in plant growth, development and stress tolerance” were reviewed in this paper. And the values of melatonin in horticultural industry were also forecasted. This review presented a summary of the investigations in the plant melatonin field, and the potential functions of increasing melatonin content in horticultural crops were also predicted.

Analysis and Comprehensive Evaluation of the Quality of Wolfberry (Lycium L.) Fresh Fruits with Different Fruit Colors

ZHAO JianHua, SHU XiaoYing, LI HaoXia, ZHENG HuiWen, YIN Yue, AN Wei, WANG YaJun

Scientia Agricultura Sinica. 2017, 50(12):  2338-2348.  doi:10.3864/j.issn.0578-1752.2017.12.014

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【Objective】The objective of this study is to explore the relations between the fresh fruit quality (FFQ) indices of wolfberry (Lycium L.) and different fruit colors and to set up a comprehensive evaluation system for wolfberry FFQ, thus would provide a theoretical basis for fresh fruit materials evaluation and utilization of wolfberry germplasm resources.【Method】Twenty-four indices of FFQ were determined in 32 wolfberry materials with different fruit colors. Eight FFQ indices were chosen by correlation analysis and factor analysis, and the weight of indices were decided by analytical hierarchy process. A comprehensive evaluation system was established for wolfberry FFQ by using data standardization process.【Result】Great differences of FFQ variations were observed in different fruit colors wolfberry, and their coefficients of variation (CV) were from14.3% to 113.4%, among which, the CV of malic acid was the highest, the CV of sucrose and flavonoids was the second, followed by the CV of cheek diameter, and total sugar was lower. Eight typically indices including length diameter (LD), cheek diameter (CD), fructose (FR), glucose (GL), oxalic acid (OC), tartaric acid (TA), flavonoids (FL) and polysaccharide(PO) were screened from 24 wolfberry FFQ indices by correlation analysis and factor analysis. An analytical hierarchy model was proposed based on the effect of eight indices on wolfberry FFQ, and weight coefficient of these factors were 17.74%, 17.74%, 10.75%, 10.75%, 5.38%, 5.38%, 10.75% and 21.51%, respectively. The values of comprehensive evaluation in tested materials were calculated according to the formula of standardized treatment, comprehensive evaluation of FFQ showed a significant difference among different fruit colors wolfberries, and with the following FFQ order: red fruit > purple fruit > yellow fruit > dark red fruit > black fruit. 【Conclusion】 FFQ of different fruit color wolfberries could be evaluated by LD, CD, FR, GL, OA, TA, FL and PO. As for comprehensive quality, red fresh fruits are the most excellent, and black fresh fruits are poor. The key factors affecting wolfberry FFQ are as follows: yield factor, flavor factor and functional active factor.

FOOD SCIENCE AND ENGINEERING

Effects of Ultrasound on Chemical Forces and Water Holding Capacity Study of Heat-Induced Myofibrillar Protein Gel

WANG JingYu, YANG YuLing, KANG DaCheng, TANG XiaoZhi, ZHANG Xing, MA Yun, NI WenXi

Scientia Agricultura Sinica. 2017, 50(12):  2349-2358.  doi:10.3864/j.issn.0578-1752.2017.12.015

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【Objective】 The influences of ultrasound on chemical forces and water holding capacity of myofibrillar protein (MP) gel were studied. The relationship between MP gel chemical forces and WHC was revealed.【Method】AA type broilers were slaughtered. The MP was extracted from breast muscle. The MP solution and heat induced gel in different ultrasound times were prepared. Total sulfhydryl group (SH) content changes of gel were used to reflect the formation of disulfide bond. Surface hydrophobicity and Zeta potential value of the gel were employed to present the hydrophobic interaction and electrostatic repulsion, respectively. The ratio of I₈₅₀/I₈₃₀ was used to show the hydrogen bonding changes of gel. Water holding capacity (WHC) of MP gel was calculated by high-speed centrifuge.【Result】As the ultrasonic time (UT) increased (0-6 min), the total SH contents of the MP  gel decreased and reactive SH content significantly increased, at stronger UT (6-15 min), the total SH and reactive SH contents all decreased. At short time (0-6 min), the changing trends of MP and MP gel about the total SH and reactive SH content were the same, while the total SH and reactive SH contents of MP all had no significant changes (P＞0.05) over 6 min, suggesting that UT promoted internal SH of MP into disulfide bond for shorter time, and UT and heating gel for longer time work together to promote reactive SH into disulfide bond. Surface hydrophobicity of MP gels increased from 1 194.1 to 1 489.5 (P＜0.05) and decreased from 1 489.5 to 1 230.8 at last, for the cavitation phenomenon induced by UT could bring the hydrophobic regions into the surface, while at UT＞6 min, the hydrophobic group was wrapped in the gel network. Zeta potential value of the gel changed from 6.03 to 7.68（P＜0.05）（0-6 min）, and then decreased over 6 min, which showed that ultrasonic wave made protein molecular chain unfolding, negatively charged by exposure, resulting in the stronger electrostatic repulsion, while excessive expansion of MP molecules were not conducive to the electrostatic force of the gel. After moderate UT (0-6 min), the normalized intensity of I₈₅₀/I₈₃₀ ratio increased from 0.9805 to 1.023 (P＜0.05), which indicated that more phenolic hydroxyl groups of tyrosine were exposed to the aqueous environment, generating hydrogen bonds with water molecules, while the ratio decreased at UT＞6 min, hydrogen bonds between water molecules and protein were weaken. At UT ≤6 min, the WHC of samples increased sharply from 47.5899% to 72.9855% (6 min) (P＜0.05), while at 9 min and above, WHC dropped gradually to 44.356%. Results of the correlation analysis showed that WHC of MP gel was unrelated to total SH content (P＞0.05) and was significantly related to reactive SH content (P＜0.05) and had a significantly correlation with Zeta potential absolute value, the surface hydrophobicity and hydrogen bonding (P＜0.01).【Conclusion】Ultrasound has significant impacts on both chemical forces and water holding capacity of the MP gel. Hydrophobic force, electrostatic repulsion and hydrogen bonding play important roles in holding water in MP gels, disulfide bonds not. Thus a value of 6 min is the optimum time for the water holding capacity of MP gel, under this condition, hydrophobic force, hydrogen bonding and electrostatic repulsion of the gel are maximum, resulting in the gel network structure of uniform density, and retaining water at best.

ANIMAL SCIENCE·VETERINARY SCIENCERE·SOURCE INSECT

Association Between SNPs in the CDS Regions of CXCR1 Gene and the Clinical Mastitis and Lifetime for Chinese Holstein

WANG MengQi, NI Wei, ZHANG HuiMin, YANG ZhangPing, WANG XiPu, JIANG YanSen, MAO YongJiang

Scientia Agricultura Sinica. 2017, 50(12):  2359-2370.  doi:10.3864/j.issn.0578-1752.2017.12.016

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【Objective】 The objective of this study was to investigate the association between the SNPs in the coding sequence (CDS) of CXCR1 gene and the clinical mastitis and lifetime for Chinese Holstein.【Method】SNPs in the CDS of CXCR1 gene were screened by using PCR and direct sequencing for 20 cows with low SCS and 20 cows with high SCS. Finally, the selected SNPs of CXCR1 gene for 866 Chinese Holstein cows were detected using flight mass spectrometry. The clinical mastitis and productive life of tested cows were collected from the management system of dairy farm. The association between the SNPs and the clinical mastitis and productive life of tested cows was analyzed using multi factor variance analysis, Logistic regression, Cox regression.【Result】A total of 13 SNPs were found in the CDS region of CXCR1 gene (291 C＞T, 333 C＞G, 337 A＞G, 365 C＞T, 570 A＞G, 642 A＞G, 735 C＞G, 816 A＞C, 819 A＞G, 980 A＞G、, 995 A＞G, 1008 C＞T and 1068 A＞G), and these SNPs were divided into 4 linkage groups, and four SNPs were selected from each linkage group for further analysis（642 A＞G, 816 A＞C, 980A＞G and 1068 A＞G). A total of 9 haplotypes were observed for 4 SNPs of CXCR1 gene, the GAGG haplotype frequency was the highest (0.3141), and the frequency of haplotype ACAA was the lowest (0.0017). CXCR1-642 A＞G showed a significant association with the number of dairy cows suffering from clinical mastitis in second lactation (P＜0.05). The number of dairy cows suffering from clinical mastitis for individual with AG genotype was significantly higher than that of AA genotype (P＜0.05). The number of dairy cows suffering from clinical mastitis for individual with CXCR1-816 AA genotype was significantly higher than that of AC and CC genotypes (P＜0.05). The other SNPs showed no significant association with the number of clinical mastitis. CXCR1-816 A＞C mutation showed an extremely significant association with culling age (P＜0.01) and a significant association with productive month and culling lactation (P＜0.05). The productive month, culling age and culling lactation for the individual of CXCR1-816 AA were significantly higher than that of CC genotype (P＜0.05). Cox regression showed that only CXCR1-816 showed significant association with the productive life（P＜0.05), the survival probability of individual with CC genotype was significantly lower than that of AA and AC genotype in each time period. 【Conclusion】The mutation of CXCR1-816 A＞C showed significant association with the clinical mastitis and lifetime for Chinese Holstein, and this SNP could be used for molecular marker-assisted selection of productive life for Chinese Holstein when the molecular function of this mutation were proved.

Validation of Candidate Gene FREM1 of H Locus of Huoyan Trait in Goose

YU JinCheng, YU Ning, ZHAO Hui, LI Zhe

Scientia Agricultura Sinica. 2017, 50(12):  2371-2379.  doi:10.3864/j.issn.0578-1752.2017.12.017

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【Objective】Recessive Z-link inheritance of the Huoyan trait has been confirmed, but its genetic basis still has not been defined. Recessive mutations in the coding region of FRAS-related extracellular matrix1 (FREM1) gene have been shown to cause partial or whole loss of upper eyelid in human and model mice. In this study, cloning, expression and polymorphism analysis of the goose FREM1 gene were carried out using goose resource populations with Huoyan trait as main materials, to verify the hypothesis that FREM1 is a candidate gene affecting eyelid traits of goose, which will lay a foundation for further study of molecular genetic mechanism of eyelid traits.【Method】The coding region of goose FREM1 gene was amplified and cloned from total RNA of goose upper eyelids and kidney tissues, which were collected from the Huoyan goose (3 females), Sichuan white goose(3males and 3females), F₁ population of male Huoyan goose mated with female Sichuan White goose(3males), byRT-PCR according to the full-length transcript of FREM1 (XM_013193557), and the bioinformation analysis was performed with it. Then, the expression of FREM1 gene in goose eyelid and kidney tissue was studied by real-time fluorescence quantitative PCR. The whole blood DNA was extracted from 30 adult male Huoyan geese, 30 male Sichuan white geese, and 30 male geese of F₁ population of Huoyan goose mated reciprocally with female Sichuan White goose, and the distribution of FREM1 gene mutations in different goose populations was detected by direct sequencing according to the genomic sequences of FREM1 (Anser cygnoides domesticus breed Zhedong scaffold203).【Result】①The 7 305bp cDNA sequences of FREM1 gene was obtained by sequencing and splicing, which contained a complete CDS region encoding 2 184 amino acids. Compared with the nucleotide sequences of the Sichuan White goose and Zhedong White goose, the c.4 515T＞ C missense mutation was found in CDS of FREM1 gene of Huoyan goose, which causes an Valine to Alanine change (p.Val1505Ala) at a highly conserved amino acid residue in FREM1’s tenth CSPG domain. Although, the p.1 505Val＞Ala change was predicted to be “Tolerated” by SIFT, it was subsequently predicted to lead to decreased protein stability by the I-Mutant ΔΔG and MUPro program. ②FREM1 gene was expressed in the tissues of eyelids and kidneys of Sichuan White goose, but the expression level in kidney was much higher than eyelid, more importantly, the expression level of the male is nearly twice as that of the female. There was no significant difference in the relative expression of FREM1 gene between Z^HW(Normal eyelids)and Z^hW(Upper eyelids colomobas) genotypes (P＞0.05). Although the relative expression of FREM1 gene of Z^HZ^h(Normal eyelids) genotype was more than 2 times than that of Z^HW and Z^hW genotype, it could be caused by differences in gender. ③In Huoyan Goose populations, the frequencies of HH, Hh and hh genotypes were 0, 0 and 1.0, the frequencies of alleles H and h were 0 and 1.0, the heterozygosity was 0; The frequencies of HH, Hh and hh were 1.0, 0 and 0 in Sichuan white goose population, the frequencies of the alleles H and h were 1.0 and 0, the heterozygosity was 0 in Sichuan White Goose populations; The frequencies of HH, Hh and hh were 0, 1.0 and 0, the frequencies of the alleles H and h were 0.5 and 0.5, the heterozygosity was 1.0 in F₁ generation population of Huoyan Goose mated reciprocally with female Sichuan White Goose.【Conclusion】The FREM1 gene is the H locus which determines the eyelid traits of goose. A homozygous missense mutation in the coding region of the FREM1gene caused an Valine to Alanine change (p.Val1505Ala) at a highly conserved amino acid residue in FREM1’s tenth CSPG domain, which affected the stability of FREM1 protein. Therefore, mutation c.4514T＞ C of gene FREM1can be used as an important molecular marker.

Comparison of Different Cold Resistance betweenApis cerana cerana and Apis mellifera ligustica

QIN Ming, WANG HongFang, LIU ZhenGuo, WANG Ying, WANG Shuai, CHI XuePeng, LIU ChunLei, ZHANG WeiXing, XU BaoHua

Scientia Agricultura Sinica. 2017, 50(12):  2380-2388.  doi:10.3864/j.issn.0578-1752.2017.12.018

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【Objective】 the objective of this research is to study the composition of cold resistance system in Apis cerana cerana and Apis mellifera ligustica, and to provide a theoretical basis for the study of the differences of cold resistance between the two species. 【Method】Five A. c. cerana colonies and five A. m. ligustica colonies which overwintered under the same equivalent environment were randomly selected with 10-25 bees in each colony on December 20th, 2015. The supercooling point (SCP) and freezing point (FP) were measured immediately, and their cold tolerance were compared. The free water, bound water, fat, glycogen, protein and the trehalase activities were determined, respectively. Four important regulatory genes were sure through the biosynthetic pathway of trehalose. The relative expression of the four important regulatory genes at mRNA level were detected by real-time fluorescent quantitative PCR in over-wintering period. 【Result】 Compared with A. m. ligustica, the super-cooling point was significantly higher than A. c. cerana (P＜0.05). The freezing point showed no obvious difference (P＞0.05). The free-water content of A. c. cerana was significantly lower than A. m. ligustica (P＜0.05), by contrast the bound-water content of A. c. cerana was obviously higher than A. m. ligustica (P＜0.05). There was no obvious difference in the fat content between A. cerana cerana and A. m. ligustica (P＞0.05). There were no significant differences in the protein content of two bee species (P＞0.05); glycogen content of A. c. cerana had significantly increased than A. mellifera ligustica in mid-winter (P＜0.05). The mannitol, sorbic alcohol content of A. c. cerana was twice as that of A. m. ligustica in the hemolymph (P＜0.05). The content of trehalose in A. c. cerana was obviously higher than A. m. ligustica (P＜0.05). The trehalase activity of A. c. cerana significantly reduced compared with A. m. ligustica in the body and hemolymph (P＜0.05). Compared with A. c. cerana, the relative expression levels of Tre-2 and UGP had significantly increased in A. m. ligustica (P＜0.05), but Tre-1 was obviously lower (P＜0.05). There was no obvious difference in the relative expression levels of TPS between A. c. cerana and A. m. ligustica (P＞0.05).【Conclusion】A. c. cerana decreases supercooling point by reducing the content of free water, increasing the content of glycogen and low molecular sugar, regulating trehalase activity related genes expression to increase its cold resistance.

RESEARCH NOTES

The Response to Heat and Screening of the Interacting Proteins of Zinc Finger Protein GmDi19-5 in Soybean

ZHAO JuanYing, LIU JiaMing, FENG ZhiJuan, CHEN Ming, ZHOU YongBin, CHEN Jun, XU ZhaoShi, GUO ChangHong

Scientia Agricultura Sinica. 2017, 50(12):  2389-2398.  doi:10.3864/j.issn.0578-1752.2017.12.019

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【Objective】Heat stress has become one of main abiotic stresses that threaten plant growth and development. Transcription factors play important roles in plant abiotic stress responses. Drought-induced protein Di19 play an important role in stress signal transduction process in plant. To further explore the functional mechanism of GmDi19-5, the function was identified in high temperature stress and its interacting proteins were screened by yeast two-hybrid system. 【Method】The real-time PCR was used to analyze the expression patterns of GmDi19-5 under high temperature stress treatment in different time periods. The 1,206 bp sequence upstream of GmDi19-5 was analyzed in PlantCARE and PLACE database. The promoter activity of GmDi19-5 was also analyzed using GUS staining in transgenic Arabidopsis. GmDi19-5 was overexpressed in Arabidopsis to statistically analyze its seed germination and root length under high temperature stress conditions. Bait plasmid pGBKT7-GmDi19-5 was constructed and the self-activation was detected. To obtain the candidate proteins of GmDi19-5, the mixture of recombinant plasmid pGBKT7-GmDi19-5, pGADT7 with soybean cDNA library was introduced into yeast cell AH109. The yeast two-hybrid system was used to screen interactive candidate proteins. Furthermore, the interaction between GmDi19-5 and candidate proteins was analyzed using the yeast two-hybrid system. The response of candidate genes to heat stress was examined by Real-time PCR; GmDi19-5was fused with GFP to detect its subcellular localization in protoplast cells of Arabidopsis. 【Result】The expression pattern analysis showed that GmDi19-5 was involved in responses to abiotic stresses and it was induced by high temperature stress in soybean. After high temperature stress treatment, GUS activities driven by promoter significantly increased in roots, leaf primordium and young leaves in transgenic Arabidopsis. PlantCARE and PLACE database analysis showed that the GmDi19-5 promoter contained a series of cis-element, including heat shock response element (HSE). The subcellular localization analysis revealed that green fluorescence of GFP-GmDi19-5 was mainly in the nucleus. The seed germination and root length assays revealed that GmDi19-5 transgenic Arabidopsis increased sensitivity to high temperature stress. Yeast two-hybrid results showed that candidate protein GmDnaJ interacted with GmDi19-5. Real-time PCR showed that the interactive candidate gene GmDnaJ was induced by heat stress. 【Conclusion】The expression of GmDi19-5 was induced by high temperature stress and its overexpression inhibited the growth and development of transgenic plants. Yeast two-hybrid analysis showed that GmDnaJ maybe interact with GmDi19-5 in yeast cells. This result suggests that GmDi19-5 maybe function in abiotic stress responses by interacting with GmDnaJ.

Reference Genes Selection and System Establishment for Real-time qPCR Analysis in Ping’ou Hybrid Hazelnut (C. heterophylla Fisch. × C. avellana L.)

YANG Dan, LI Qing, WANG GuiXi, MA QingHua, ZHU LiQuan

Scientia Agricultura Sinica. 2017, 50(12):  2399-2410.  doi:10.3864/j.issn.0578-1752.2017.12.020

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【Objective】 The objective of this article is to construct a reference gene screening system of real-time qPCR in Ping’ou hybrid hazelnut (C. heterophylla Fisch × C. avellana L, main cultivars of Corylus in China) for gene expression analysis, and provide a theoretical basis for the study of plant resource utilization and innovative breeding. 【Method】 Eight candidate genes were selected from the transcriptome sequencing data of the non-pollination, compatible pollination and incompatible pollination stigma of Ping’ou hybrid hazelnut in authors’ previous study. Four candidate genes were selected from related articles. Eight different tissues or organs, such as the blooming styles, the catkins before elongation, the young leaves, the pollen, the cambium of annual branch, the green stem, the root tip and the sucker of the main cultivar of Ping’ou hybrid hazelnut ‘Dawei’ were used as the samples in reverse transcriptional PCR and real-time qPCR experiments. The expression stability of twelve candidate reference genes was analyzed by geNorm, NormFinder, BestKeeper, Delta Ct and RefFinder programs. 【Result】 Reverse transcriptional PCR showed that the amplification of twelve primers was specific, there were significant differences in the expression of ChaSTP5 and ChaTF in different materials, and the remaining candidate reference genes were expressed in eight tissues. Real-time qPCR showed that the expression of Ch18S rRNA was at the highest level and ChaSTP5 was at the lowest level, and the remaining ten candidate reference genes belong to moderate expression. As for the stability of the candidate genes, ChaSTP5 and ChaTF were the least stable, and the stability of the remaining ten candidate genes was at a moderate level. The results of geNorm, NormFinder, BestKeeper and Delta Ct showed that ChaActin was the most stable reference gene and Ch18S rRNA ranked in the top five, while the ranking of other candidate reference genes was different. The stability analysis indicated that ChaActin and Ch18S rRNA are suitable as reference genes. The pairwise variation (V) calculated by geNorm showed that six reference genes could accurately normalize the date of real-time qPCR. There was a significant correlation between the four programs at 0.01 level, and the correlation between NormFinder and Delta Ct was the highest, Delta Ct and BestKeeper was the lowest. 【Conclusion】 The reference gene screening system for real-time qPCR in Ping’ou hybrid hazelnut was set up including four main steps: primers were screened first by reverse transcriptional PCR, real-time qPCR analysis was based on primer properties and gene expression, primer stability was evaluated using four programs (geNorm, NormFinder, BestKeeper, Delta Ct) and the optimal stable reference gene was selected by the comprehensive analysis of RefFinder. As for the reference genes selection, ChaActin and Ch18S rRNA were ranked as the most stable reference genes in 8 samples.