一种快速、无损大豆种子DNA提取方法的建立和应用

基因分型是进行植物基因功能的遗传分析和分子标记辅助育种的重要环节。该研究以大豆(Glycine max)成熟种子为材料, 建立了通过钻孔采集样品、快速提取DNA进行基因型鉴定的方法。用此方法, 一个熟练的工作人员可以在1个小时内完成120个样品的采集和DNA提取; 同时种子钻孔取样后, 不会对大豆种子的萌发造成影响。利用该方法获得的DNA可满足PCR扩增的要求。实验重复性好, 成功率在98%以上。这种快速且无损的大豆种子基因型鉴定方法可以用于鉴定杂交种子、品种纯度以及遗传分析等研究工作。     

高效液相色谱(HPLC)技术鉴定中国南方大豆品种异黄酮主要组分

采用高效液相色谱(HPLC)技术检测中国南方六个省份的249份大豆品种异黄酮主要组分含量.结果显示大豆籽粒中可检测出6种主要的异黄酮组分,分别为大豆甙(Daidzin)、甲氧基黄豆甙原(Glycitin)、染料木甙(Genistin)、丙二酰基大豆甙(Malonyldaidzin)、丙二酰基黄豆甙原(Malonylglycitin)和丙二酰基染料木甙(Malonylgenistin).各组分中以丙二酰基(Malonyl)异黄酮组分含量最高(61.2%),且各组分间相关极显著.大豆品种间异黄酮含量变异较大,变异系数达49.6%.来自江苏省的品种海门红黄豆乙异黄酮含量最高(4932.3μg/g),品种宝应等西风含量最低(367.1μg/g).不同省份间异黄酮含量差异极显著,来自浙江省的大豆品种平均含量最高(2717.2μg/g),来自安徽省的平均含量最低(1181.8μg/g).异黄酮含量与生育期呈极显著正相关(r=0.319* * *),与百粒重呈显著正相关(r=0.132*),而与脂肪含量(r=-0.45* * *)和蛋白质含量(r=-0.136)呈负相关.     

微量大豆种子基因组DNA的快速制备

用改良的CTAB法,从微量大豆种子样品中快速提取了基因组DNA,并从大豆基因组中扩增到了大豆蛋白酶抑制剂基因.     

一个种子特异的大豆AP2类转录因子参与调控种子萌发

植物种子的发育和萌发受严格的时空调控,其中转录因子起着重要的作用．本研究发现了一条在发育过程中的大豆种子里特异表达的表达序列标签（expressed sequence tag,EST）．通过末端快速扩增技术（rapid amplification of cDNA end,RACE）得到该基因的全长序列并命名为GmSGR．序列分析表明该基因属于AP2／ERF类转录因子家族,其AP2结构域与拟南芥（Arabidopsis thaliana）中的AP2／ERF基因家族DREB亚家族中A-3组的AtABI4的AP2结构域同源性极高．在酵母系统中,未检测到GmSGR具有转录激活活性．将该基因在拟南芥中过量表达,在高浓度的脱落酸（abscisicacid,ABA）及葡萄糖的条件下,转基因植物种子的萌发率均高于对照组;而在高浓度的NaCl条件下,转基因植物种子的萌发率低于对照组．在转基因植物的幼苗中,AtEm6和AtRD29B的表达较野生型幼苗高．这些结果表明GmSGR可能通过调控AtEm6和AtRD29B的表达,从而导致转基因种子对ABA敏感度降低,对盐胁迫更敏感．     

Quantitative Trait Locus Mapping of Seed Vigor in Soybean under −20 °C Storage and Accelerated Aging Conditions via RAD Sequencing

Due to its fast deterioration, soybean (Glycine max L.) has an inherently poor seed vigor. Vigor loss occurring during storage is one of the main obstacles to soybean production in the tropics. To analyze the genetic background of seed vigor, soybean seeds of a recombinant inbred line (RIL) population derived from the cross between Zhonghuang24 (ZH24, low vigor cultivar) and Huaxia3hao (HX3, vigorous cultivar) were utilized to identify the quantitative trait loci (QTLs) underlying the seed vigor under −20 °C conservation and accelerated aging conditions. According to the linkage analysis, multiple seed vigor-related QTLs were identified under both −20 °C and accelerated aging storage. Two major QTLs and eight QTL hotspots localized on chromosomes 3, 6, 9, 11, 15, 16, 17, and 19 were detected that were associated with seed vigor across two storage conditions. The indicators of seed vigor did not correlate well between the two aging treatments, and no common QTLs were detected in RIL populations stored in two conditions. These results indicated that deterioration under accelerated aging conditions was not reflective of natural aging at −20 °C. Additionally, we suggest 15 promising candidate genes that could possibly determine the seed vigor in soybeans, which would help explore the mechanisms responsible for maintaining high seed vigor.     

Isolation and Partial Characterization of the Major Seed Protein from Nicotiana tabacum,and Accumulation during Development

During the seed development of Nicotiana tabacum, appreciable accumulation of the soluble protein fraction started to occur at around the 6th day after anthesis and finally reached 12% on the basis of dry weight when seed maturation was accomplished. In the soluble fraction of mature seeds, four protein fractions were observed on analytical ultracentrifugation, and the protein having a sedimentation coefficient of 11.7S was the major one. The 11.7S protein was isolated and SDS-polyacrylamide gel electrophoresis indicated that the protein consisted of at least five subunits with molecular weights of 49,000, 31,000, 29,000, 21,000 and 19,000. The 11.7S protein was rich in glutamic acid or glutamine and arginine, and the presence of carbohydrate was confirmed.

During development, all of the five subunits started to appear during the period between the 12th and 15th day after anthesis.     

菜用大豆种子活力与DNA,RNA及蛋白质合成的关系

菜用大豆种子随着其活力的下降,对DNA,RNA和蛋白质前体的吸收,以及合成这些大分子的能力都明显下降,已丧失合成DNA和蛋白质能力的失活种子,仍能进行微弱的RNA合成。高活力种子在吸胀初期DNA合成速率较低,然后增加,至16h达高峰;RNA的合成速率在吸胀一开始就很高,在整个吸胀过程中均保持较高水平;蛋白质的合成速率则在开始较高,并随着吸胀过程呈增强趋势。     

大豆籽粒发育过程中异黄酮合成相关酶基因的表达模式与异黄酮积累的相关分析

利用高效液相色谱法和实时定量PCR方法,分别测定了2个异黄酮含量显著差异的大豆品种鲁黑豆2号(LHD2)和南汇早黑豆(NHZ)在子粒发育过程中的异黄酮含量变化以及异黄酮合成相关酶基因的表达模式变化,试图分析异黄酮积累与各基因表达量变化的相关关系。结果表明在大豆子粒发育过程中,异黄酮含量逐渐升高,而不同异黄酮合成相关酶基因的表达趋势不同,CHS7、CHS8、CHR、CHI1A和IFS2的表达趋势与异黄酮积累模式基本一致,而IFS1和CHI1B1的表达趋势与异黄酮积累模式相反。IFR的表达模式在2个大豆品种中存在相反的趋势,在LHD2中与异黄酮组分积累趋势相反,而在NHZ中与异黄酮组分积累趋势相同。结果还表明,同一基因家族中不同基因在子粒发育过程中的表达量也存在差异。查尔酮合酶基因家族中CHS7和CHS8以及查尔酮异构酶基因家族的CHI1A的表达水平相对其他成员较高,异黄酮合酶基因家族中IFS2的表达量显著高于IFS1的表达量,预示这些基因家族在大豆子粒异黄酮积累过程中存在功能分化。此外,各基因表达模式与异黄酮积累的相关分析结果表明,不同基因表达模式与异黄酮积累的相关性在2个品种中也不尽相同。LHD2中CHS7、CHS8和IFS2在子粒发育过程中的表达量变化与不同异黄酮组分呈显著正相关,CHI1B1基因的表达量变化与不同异黄酮组分呈显著负相关。而在NHZ中,IFR在子粒发育过程中的表达量变化与多个异黄酮组分呈显著正相关。这预示了不同大豆品种异黄酮含量差异的潜在遗传基础。各异黄酮合成相关酶基因表达量变化的相关分析表明,在2个品种中,苯丙氨酸水解酶PAL1与4CL,4CL与CHS2以及CHS1与IFS2基因的表达量均呈现显著正相关。表明这些基因可能通过协同作用共同调控异黄酮的合成与积累。这些结果为今后利用基因工程提高大豆异黄酮含量奠定了基础。     

A Rapid DNA Extraction Method for RFLP and PCR Analysis from a Single Dry Seed

A single-seed DNA extraction method was developed for rapid identification of plant genotype. The method was applied to 12 plant species, including the oil seeds sesame and soybean. The results were comparable to those obtained for oil-less seeds such as rice. This method will be useful for genotypic selection which requires rapid screening of large populations. It can also be used to identify varietal purity of seed stocks by PCR and RFLP analysis. The method includes two major steps, (i) treatment by proteinase K in an SDS extraction buffer, and (ii) grinding of a single half seed in the buffer after incubation. About 1.5–2 µg of DNA per half seed (the endosperm part) of rice was obtained and more than 200 half seed samples could be handled by one person in a day. The DNA could be used for fingerprinting and detection of target genes in a transgenic plant by PCR. The amplified PCR products from the half seed DNA exhibited the same banding patterns as those from leaf DNA. Yield and quality of DNA extracted from half seeds of rice was also sufficient for RFLP analysis. The remnant half seeds containing the embryo can be maintained for later germination of selected genotypes.     

Phosphorus and Nitrogen Interactions in Field-Grown Soybean as Related to Genetic Attributes of Root Morphological and Nodular Traits

Two field experiments with different soybean (Glycine max L.) materials were conducted to investigate the interactions between phosphorus (P) and nitrogen (N) as related to the genetic attributes of root morphological and nodular traits. In experiment one, 13 cultivated soybean varieties were grown in a field with relatively low soil P and N availability. P application with 160 kg P/hm2 as triple superphosphate produced a significant simultaneous increase in the content of both P and N in shoot, demonstrating positive P and N interactions. The addition of P also increased root dry weight, root nodule number, nodule mass, nodule size, and nodulation index, but decreased root length and root surface area, indicating that P may affect N nutrition in soybean through a number of root morphological and nodular traits. Interestingly,like P content, N content appeared to be more correlated with root morphological traits (root weight, root length, and root surface area) than with root nodular traits (nodule number, nodule size, nodule mass, and nodulation index) at both P levels, implying that N taken up by the roots may contribute more to the plant N status than biological N2 fixation under the present experimental conditions. In experiment two, 57 soybean lines of a recombinant inbred line (RIL) population derived from a cross between a cultivated variety and a wild genotype were grown on another field site with moderately sufficient P and N levels to further characterize the genetic attributes of root morphological and nodular traits and their relationships with P and N interactions. The results indicated that all morphological and nodular traits measured continually segregated in the RIL population with a normal distribution of the phenotypic values, indicating that these traits are possibly controlled by quantitative trait loci (QTLs). Genetic analysis revealed that all these root traits had relatively low heritabilities (h2b=74.12, 70.65, 73.76, 56.34, 52.59, and 52.24 for root weight, root length,root surface area, nodule number, nodule mass, and nodule size, respectively), suggesting that root morphology and nodule formation are influenced greatly by environmental factors. Correlation analysis of the RILs showed that shoot N content was significantly correlated with P content, confirming positive P×N interactions. Similar to experiment one, shoot N content was only significantly correlated with root morpho logical traits, but not with root nodular traits, again denoting the fact that the N status in soybean could be attributed more to N uptake from the soil than to biological N2 fixation under the present experimental conditions.     

Superoxide Dismutase (SOD)Zymogram Patterns and Their Geographical Distribution of Wild (Glycine soja) and Cultivated Soybean (G. max) in China

The purpose of this study was to examine the superoxide dismutase (SOD) zymogram patterns, their frequency and geographical distribution of wild (Glycine soja) and cultivated soybean (G. max) in China. Seeds of 226 wild soybean germplasms and 104 cultivated soybean cultivars (land races) were collected from all provinces and autonomous regions in China except Taiwan, Xinjiang and Qinghai provinces About 50 embryos per wild soybean germplasm and I0 embryos per cultivated soybean cultivars were used for test. Vertical polyacrylamide gel electrophoresis and a stainning system modified after Luo (1984)were used. The Japanese GS- 930 Scanner was used in gel-plate scanning. In program scanning the maximum and minimum absorption wavelength were 700 and 550 nm respectively. The results showed that: 1. Six zymogram patterns were found in soybean (Fig. 1, 2). Wild soybean displayed five patterns (Ⅰ, Ⅱ, Ⅳ Ⅴ, Ⅵ), while the cultivated soybean displayed only two patterns (Ⅱ, Ⅲ). 2. Fourty six percent of wild germplasms gave an 7-band zymogram (Table Ⅰ) (pattern Ⅰ), fourty nine percent had a 6th and 7th band with faster mobility (pattern Ⅱ), about two percent produced a 6-band zymogram which lacked the SODc4 band (pattern Ⅳ), about two percent had a 5-band pattern which lacked the SODc,c4 bands (pattern Ⅴ), and only one germptasm displayed a 5-band zymogram which lacked SODb2b3 bands (pattern Ⅵ). 3. More than ninty eight percent of cultivated cultivars belonged to pattern Ⅱ, only about two percent belonged to pattern Ⅲ. 4. The geographical distribution of frequency of pattern Ⅱ between wild and cultivated soybean was most close in 36–51º N area. The difference of zymograms between G. soja and G. max, and the problems of the origional area and evolution of soybean were discussed.     

基于小波分析的大豆叶绿素a含量高光谱反演模型

 2003和2004年分别在长春市良种场和中国科学院海伦黑土生态实验站实测了大田耕作与水肥耦合作用下大豆（Glycine max）冠层高光谱反射率 与叶绿素a含量数据,对光谱反射率、微分光谱与叶绿素a含量进行了相关分析;采用归一化植被指数(Normalized diffe rence vegetation index, NDVI)、土壤调和植被指数(Soil-adjusted vegetation index, SAVI)、再归一植被指数(Renormalized difference vegetation index, RDVI)、第二修正比值植被指数(Modified second ratio index, MSRI)等建立了大豆叶绿素a反演模型;应用小波分析对采集的光谱反 射率数据进行了能量系数提取,并以小波能量系数作为自变量进行了单变量与多变量回归分析,对大豆叶绿素a进行了估算。研究结果表明,大 豆叶绿素a 与可见光光谱反射率相关性较好,并在红光波段取得最大值(R²>0.70),但在红边处,微分光谱与大豆叶绿素a的相关性较反射率好 得多,在其它波段则相反;由NDVI、SAVI、RDVI、MSRI等植被指数建立的估算模型可以提高大豆叶绿素a的估算精度（R²>0.75）;小波能量系 数回归模型可以进一步提高大豆叶绿素a含量的估算水平,以一个特定小波能量系数作为自变量的回归模型,大豆叶绿素a回归决定系数R²高达 0.78;多变量回归分析结果表明,大豆叶绿素a实测值与预测值的线性回归决定系数R²均高达0.85。以上结果表明, 小波分析可以对高光谱进 行特征变量提取,并可在一定程度上提高大豆生理参数反演精度。