小麦高分子量谷蛋白亚基Glu-B1位点沉默基因的克隆与序列分析

二粒小麦（Triticum turgidum L．var．dicoccoides）具有极其丰富的遗传多样性,是栽培小麦品种改良的巨大基因库。在高分子量谷蛋白基因的组成上,它具有许多栽培小麦不存在的变异类型,在Glu—B1位点上的变异更大。我们利用种子贮藏蛋白的SDS—PAGE方法从原产于伊朗的二粒小麦材料PI94640中观察到缺失Glu—B1区的高分子量谷蛋白亚基。利用Glu-1Bx基因保守序列设计PCR引物,对该材料的总DNA扩增,获得了X型亚基编码基因（Glu-1Bxm）的全序列,其全长为3442bp含1070bp的启动子区。序列比较发现,Glu-1Bxm在启动子区序列与Glu—1Bx7的最为相似。而在基因编码区,我们发现Glu—1Bxm仅编码212个氨基酸,由于开放阅读框中起始密码子后第637位核苷酸发生了点突变,即编码谷酰胺的CAA突变为终止密码TAA,可能直接导致了该高分子量谷蛋白亚基的失活,这是我们在小麦Glu—B1位点基因沉默分子证据的首次报道。将Glu—1Bxm全序列与Glu—B1位点其他等位基因进行了系统树分析,发现Glu—1Bxm是较为古老的类型。本文还对该特异高分子量谷蛋白亚基变异类型对品质遗传改良研究的意义进行了讨论。     

不同芸豆品种种子发育过程中贮藏蛋白积累研究

以半蔓生型奶花芸豆(Y09)和直立型奶花芸豆(Y06)为材料,通过SDS-PAGE分析研究了开花后种子形成过程中贮藏蛋白(SP)的积累规律.结果表明:2个芸豆品种开花后种子形成过程中,子叶、胚蛋白积累变化趋势基本一致,但其变化幅度存在显著差异,其中,在开花后20d内,半蔓生型芸豆Y09的籽粒蛋白积累量大于直立型芸豆Y06,但在开花后25~40d,Y06籽粒蛋白积累量显著高于Y09;两芸豆品种的子叶和胚均含有丰富的贮藏蛋白(SP)(14.4~97.4kD),其中子叶的41.0、43.9、39.4kD3种蛋白含量较高,约占子叶总蛋白含量(光密度)50%以上,而胚蛋白亚基分布相对均匀;两品种的子叶、胚中蛋白亚基差异较小,只有个别亚基存在差异.     

莲子贮存蛋白的主要亚基及积累模式

红莲（NelumbonuciferaGaertn）成熟于叶总蛋白含量达24．35g／100g干样品,其贮存蛋白（SP）的积累模式与豆科种子相似,即随成熟度的提高,SP猛增至占总蛋白含量的86％以上。对莲子不同发育阶段子叶蛋白SDS-PAGE图谱的光密度测定表明：莲子叶蛋白有12个主要SP亚基（SP1－12）。按分子量（MW）大小和积累顺序可分3组：A组是98kD和93kD的2个亚基,MW最大,积累最晚,但量最多;B组是3个亚基,MW为55－50kD,大小居中,积累最早,量最少;C组的7个亚基MW最小,在27－14kD之间,积累较早,量较多。对莲的不同品种、不同器官进行分析后发现,它们都有1个主峰为68kD的多连峰的代谢蛋白亚基,可能是莲共有的蛋白亚基。     

莲子贮存蛋白的主要亚基及积累模式

红莲(Nelumbo nucifera Gaertn)成熟子叶总蛋白含量达24.35 g/100 g干样品,其贮存蛋白(SP) 的积累模式与豆科种子相似,即随成熟度的提高,SP猛增至占总蛋白含量的86%以上。对莲子不同发育阶段子叶蛋白SDS-PAGE图谱的光密度测定表明莲子叶蛋白有12个主要SP亚基(SP 1～12)。按分子量(MW)大小和积累顺序可分3组A组是98 kD和93 kD的2个亚基,MW最大,积累最晚,但量最多;B组是3个亚基,MW为55～50 kD,大小居中,积累最早,量最少;C组的7个亚基MW最小,在27～14 kD之间,积累较早,量较多。对莲的不同品种、不同器官进行分析后发现,它们都有1个主峰为68 kD的多连峰的代谢蛋白亚基,可能是莲共有的蛋白亚基。     

水稻胚胎发育与萌发过程中凝集素的生物合成及其对转录与翻译抑制剂的敏感性

稻胚凝集素(RGL)在开花后7—13天之间合成活性很强,15天以后明显下降。7—13天之间RGL合成相当旺盛是由于这一时期编码RGL的mRNA得到迅速转录,而在开花后15—30天之间以及萌发4小时内RGL的合成主要是由胚分化发育期间(7—13天)所形成的mRNA所指导的。RGL主要在水稻胚胎发育过程中合成、积累,在萌发过程中几乎不表达,所以RGL是胚胎特异的蛋白质。     

激光诱变小麦后代材料籽粒贮藏蛋白编码基因变异的研究

本研究采用SDS—PAGE分析了冀麦七号、冀麦18和冀早15三个品种激光后代籽粒贮藏蛋白的变异情况。结果表明,高分子量麦谷蛋白和醇溶蛋白亚基变异较小,其控制位点为Glu—1和Gl—3或Gli—1;而低分子量部分变异较大,其控制位点多在Glu—3、Gli—2和Gli—3。这些变异具有一定的随机性。有关激光诱变对小麦面粉品质的影响有待进一步研究。     

草地贪夜蛾V-ATPase亚基A、B、C和D基因的克隆与分析

【目的】克隆获得草地贪夜蛾Spodoptera frugiperda V-ATP酶（Vacuolar-type proton,ATPase）V1结构域A、B、C和D亚基的cDNA序列,分析其在草地贪夜蛾不同生长阶段的表达情况,为筛选新型农药的靶点奠定基础。【方法】利用PCR技术获得草地贪夜蛾V-ATP酶V1结构域A、B、C和D亚基基因的ORF区域,采用qRT-PCR技术检测各基因在草地贪夜蛾不同发育时期的表达水平。【结果】A、B、C和D亚基基因均在不同鳞翅目昆虫中高度保守。A-D亚基在草地贪夜蛾各生长阶段均有表达,在幼虫阶段和成虫阶段的表达量均较高,表明V-ATP酶可能影响幼虫生长发育和成虫交配繁殖。卵期和蛹期表达量低,这与卵期和蛹期昆虫各项生命活动低有关。各亚基在各发育时期的表达量存在差异,说明V-ATP酶A、B、C和D发挥不同的功能。【结论】从草地贪夜蛾中克隆了V-ATP酶V1结构域A、B、C和D亚基4个基因在草地贪夜蛾不同发育阶段表达量具有差异,推测其对草地贪夜蛾的生长、发育和繁殖具有重要的调控作用。     

甘肃省春小麦品种高分子量麦谷蛋白亚基组成分析

应用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)技术,对甘肃省121个普通小麦品种(包括地方品种、育成品种和引进品种)高分子量谷蛋白亚基构成、不同等位基因间亚基变异及出现频率系统分析的结果表明,甘肃省春小麦品种HMW-GS等位基因Glu-Al编码1、2*、N(缺失)三种亚基,Glu-B1 编码 7 8、7 9、17 18、22、7、8、14 15七种亚基,Glu-D1编码2 12、2 11、5 10、2 10、10、4 11、3 12、N八种亚基,频率最高的亚基分别是Null(81.0%)、7 8(86.0%)、2 12(60.0%);一些优质亚基在现有品种中都存在,如甘春20、高台紫麦子等,作亲本用于品质改良有很大潜力,而且少数罕见亚基7、8、11也存在;不同来源的春小麦品种HMW-GS组成比较,优质亚基组成类型如1、7 8、5 10,1、17 18、5 10在育成品种中出现频率较高.     

淡色库蚊ATP合酶B亚基基因克隆和序列分析及其与溴氰菊酯抗性的关系

【目的】克隆淡色库蚊Culex pipiens pallens ATP合酶B亚基基因编码区序列,并进行生物信息学分析,研究其与溴氰菊酯抗性关系。【方法】通过PCR方法扩增ATP合酶B亚基基因编码区序列;利用生物信息学网站在线分析工具,预测ATP合酶B亚基基因编码蛋白的理化性质和功能特征;通过实时定量PCR方法比较ATP合酶B亚基基因在室内筛选的淡色库蚊溴氰菊酯敏感品系和抗性品系中的表达,并在现场种群溴氰菊酯敏感和抗性个体中做进一步验证。【结果】成功克隆淡色库蚊ATP合酶B亚基基因编码区序列(Gen Bank登录号:KY783434),长717 bp,编码238个氨基酸。生物信息学分析表明,其编码蛋白理论相对分子量为26.96 k D,等电点为8.97,在第70-231位氨基酸具有ATP合酶B亚基结构域,未发现信号肽和跨膜区。实时定量PCR结果显示,ATP合酶B亚基基因在室内筛选的淡色库蚊抗溴氰菊酯品系和现场种群溴氰菊酯抗性个体中表达量均上调。【结论】本研究获得了淡色库蚊ATP合酶B亚基基因编码区序列,进行了生物信息学分析,并证实其在溴氰菊酯抗性个体中高表达,为进一步研究该基因在蚊抗药性中的作用奠定了基础。     

气象因子对油菜种子中油分积累的影响

选取2个甘蓝型油菜(Brassica napus)高油材料XZ37(含油量45.29%)和XZ366(含油量43.48%), 分别种植在南京、拉萨和西宁, 探讨种子发育过程中油分积累的差异, 并分析南京、拉萨和西宁3个地理生态环境下油菜花–角果期间的主要气象因子与种子油分之间的相关性及其对种子油分积累的影响。结果表明, 不同生态地区间种子中油分积累差异显著。在西宁种子油分快速积累始于开花后19天, 持续时长15天; 在南京种子油分快速积累始于开花后24天, 持续时长15天; 在拉萨种子油分快速积累始于开花后29天, 持续时间长达20天。研究显示, 日均温度、日均温差、日均降水量是影响甘蓝型油菜种子发育过程中油分积累的主要气候因子。不同地理生态地区, 影响油菜种子中油分积累的主要气候因子不同。日均温度是影响南京地区种子发育过程中油分积累的主要气候因子。该地区油菜开花后, 气温由低到高呈上升趋势, 成熟后期温度偏高, 不利于种子中油分积累。日均温差和日均降水量是影响拉萨和西宁两地种子油分积累的主要气候因子。两地种子发育过程中日均温差大, 种子中油分积累量大, 但由于拉萨日均降水量高于西宁, 日均温度偏低, 种子油分积累量低于西宁。因此, 在油菜种子发育过程中, 适宜的温度、较大的温差和较少的降水有利于种子积累油分, 并形成较高的含油量。     

Allelic variation of high-molecular-weight glutenin genes in bread wheat

The composition and quantity of high-molecular-weight glutenin subunits plays an important role in determining the bread-making quality of wheat. Molecular-genetic analysis of allelic composition of high-molecular-weight glutenin genes in 102 bread wheat cultivars and lines from different geographical regions was conducted. Three alleles at the Glu-A1 locus, nine alleles at the Glu-B1 locus, and two alleles at the Glu-D1 locus were identified. Among the investigated cultivars and lines, 21 were characterized by intracultivar polymorphism. High allelic variation of high-molecular-weight glutenin subunit genes was shown for the collection: 21 and 9 combinations were defined in monomorphic and polymorphic cultivars and lines, respectively. However, the major part of the collection (66.7%) contained four allelic combinations: Glu-A1b Glu-B1c Glu-D1d, Glu-A1b Glu-B1c Glu-D1-2a, Glu-A1a Glu-B1c Glu-D1d, and Glu-A1b Glu-B1c Glu-D1d/Glu-D1-2a. Fourteen cultivars of bread wheat were selected, and they were characterized by a favorable allelic composition of Glu-1 loci.     

Biosynthesis of storage proteins in developing rice seeds

Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the starchy endosperm protein of rice (Oryza sativa L. Japonica cv Koshihikari) during seed development confirmed that storage protein begins to accumulate about 5 days after flowering. Two polypeptide groups, 22 to 23 and 37 to 39 kilodaltons, the components of glutelin, the major storage protein in rice seed, appeared 5 days after flowering. A 26-kilodalton polypeptide, the globulin component, also appeared 5 days after flowering. Smaller polypeptides (10- to 16-kilodaltons) including prolamin components, appeared about 10 days after flowering. In contrast, the levels of the 76- and 57-kilodalton polypeptides were fairly constant throughout seed development. Transmission electron microscopy and fractionation by sucrose density gradient centrifugation of the starchy endosperms at various stages of development showed that protein body type II, the accumulation site of glutelin and globulin, was formed faster than protein body type I, the accumulation site of prolamin.

The 57-kilodalton polypeptide but not the glutelin subunits was labeled in a 2-hour treatment with [¹⁴C]leucine given between 4 and 12 days after flowering to developing ears. In vivo pulse-chase labeling studies showed the 57-kilodalton polypeptide to be a precursor of the 22 to 23 and 37 to 39 kilodalton subunits. The 57-kilodalton polypeptide was salt-soluble, but the mature glutelin subunits were almost salt insoluble.

In vitro protein synthesis also showed that the mRNAs directly coding the 22 to 23 and 37 to 39 kilodalton components were absent in developing seeds and that the 57-kilodalton polypeptide was the major product. Thus, it was concluded that the two subunits of rice glutelin are formed through post-translational cleavage of the 57-kilodalton polypeptide.

     

小麦新品种(系)Glu-1位点等位基因变异研究

应用SDS-PAGE技术分析了40份小麦新品种(系)的高分子量麦谷蛋白亚基等位基因变异。在Glu-1位点共检测到10种变异类型,其中Glu-Al位点有3种类型：Null、1、26 ,Glu-B1位点有5种类型：7 8、7 9、14 15、7、17 18,Glu-D1位点有2种类型：2 12、5 10;Null(54．3％)、7 8(51．4％)和2 12(62．9％)分别是Glu-Al、Glu-B1和Glu-D1位点上的主要亚基变异类型。另外,在2份材料的Glu-B1和Glu-D1位点各检测到1个新的亚基,分别命名为1By8．1和1Dx5^ 。Glu-1位点的Nei‘s遗传变异指数平均为0,5648,Glu-B1的遗传多样性最高,Glu-D1最低。供试小麦材料Glu-1位点的HMW-GS组合共有17种类型,以(Null,7 8,2 12)组合为主要类型,占31．4％;有9种亚基组合类型分别只在1份材料中出现,占26.1％。结果表明,这些小麦新品种(系)存在着丰富的亚基组合类型。     

New DNA markers for high molecular weight glutenin subunits in wheat

End-use quality is one of the priorities of modern wheat (Triticum aestivum L.) breeding. Even though quality is a complex trait, high molecular weight (HMW) glutenins play a major role in determining the bread making quality of wheat. DNA markers developed from the sequences of HMW glutenin genes were reported in several previous studies to facilitate marker-assisted selection (MAS). However, most of the previously available markers are dominant and amplify large DNA fragments, and thus are not ideal for high throughput genotyping using modern equipment. The objective of this study was to develop and validate co-dominant markers suitable for high throughput MAS for HMW glutenin subunits encoded at the Glu-A1 and Glu-D1 loci. Indels were identified by sequence alignment of allelic HMW glutenin genes, and were targeted to develop locus-specific co-dominant markers. Marker UMN19 was developed by targeting an 18-bp deletion in the coding sequence of subunit Ax2* of Glu-A1. A single DNA fragment was amplified by marker UMN19, and was placed onto chromosome 1AL. Sixteen wheat cultivars with known HMW glutenin subunits were used to validate marker UMN19. The cultivars with subunit Ax2* amplified the 362-bp fragment as expected, and a 344-bp fragment was observed for cultivars with subunit Ax1 or the Ax-null allele. Two co-dominant markers, UMN25 and UMN26, were developed for Glu-D1 by targeting the fragment size polymorphic sites between subunits Dx2 and Dx5, and between Dy10 and Dy12, respectively. The 16 wheat cultivars with known HMW glutenin subunit composition were genotyped with markers UMN25 and UMN26, and the genotypes perfectly matched their subunit types. Using an Applied Biosystems 3130xl Genetic Analyzer, four F₂ populations segregating for the Glu-A1 or Glu-D1 locus were successfully genotyped with primers UMN19, UMN25 and UMN26 labeled with fluorescent dyes.     

Correlation between Protein Body Formation and Storage Protein Accumulation in Soybean Cotyledons

At 20 days after flowering (DAF), the 7S α' and α subunits began to accumulate. At 25 DAF, the 7Sβ, l1SA and llSB subunits appeared. Five days later, the 11SA-4 subunit was present During the period of 25–55DAF, the storage protein content continued to increase. From 55 to 63 DAF, there was a decrease in the synthetic rate of the storage proteins. Comparing these results with the two paths of protein body formation reported previously, we draw the conclusion that the protein bodies developed from vacuoles contained not only the 7S bm also the lis proteins in soybean cotyledon cells.     

Allelic variation of high molecular weight glutenin subunits of bread wheat in Hebei province of China

In common wheat (Triticum aestivum L.), allelic variations of Glu-1 loci have important influences on grain end-use quality. The allelic variations in high molecular weight glutenin subunits (HMW-GSs) were identified in 151 hexaploid wheat varieties representing a historical trend in the cultivars introduced or released in Hebei province of China from the years 1970s to 2010s. Thirteen distinct alleles were detected for Glu-1. At Glu-A1, Glu-B1 and Glu-D1, we found that the most frequent alleles were the 1 (43.0%), 7+8 (64.9%), 2+12 (74.8%) alleles, respectively, in wheat varieties. Twenty two different HMW-GS compositions were observed in wheat. Twenty-five (16.6%) genotypes possessed the combination of subunits 1, 7+8, 2+12, 25 (16.6%) genotypes had subunit composition of 2*, 7+8, 2+12; 20 (13.2%) genotypes had subunit composition of null, 7+8, 2+12. The frequency of other subunit composition was less than 10%. The Glu-1 quality score greater than or equal to 9 accounted for 20.6% of the wheat varieties. The percentage of superior subunits (1 or 2* subunit at Glu-A1 locus; 7+8, 14+15 or 17+18 at Glu-B1 locus; 5+10 or 5+12 at Glu-D1 locus) was an upward trend over the last 40 years. The more different superior alleles correlated with good bread-making quality should be introduced for their usage in wheat improvement efforts.     

Biochemical changes in developing seeds of pigeonpea (Cajanus cajan)

Soluble sugars, starch, soluble nitrogen and protein nitrogen were studied in developing seeds of 3 cultivars of pigeonpea. When expressed on a per seed basis soluble sugars increased up to 35 days after flowering and then declined slightly. Rapid starch accumulation was observed between 14 and 28 days after flowering. The levels of soluble nitrogen and protein nitrogen underwent rapid changes during the same period. Amino-acid composition of seed protein was also studied at different stages of maturation. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of salt-soluble proteins revealed that seed storage globulins are formed after 14 days of flowering and do not change much during later stages of maturation.     

High Molecular Weight Glutenin Subunit Composition of Indian Wheats and Their Relationships with Dough Strength

One hundred and seventy two wheat varieties including twenty-five durum wheat cultivars were evaluated for high molecular weight glutenin subunit (HMW-GS) composition using SDS-PAGE. The relationship between HMW-GS and sedimentation tests for dough strength was studied. Three alleles were present at the Glu-A1 locus, eight at Glu-B1 and two at Glu-D1 in bread wheat. The data indicated the prevalence of the Glu-A1b allele (63.5%) at the Glu-A1 and Glu-D1a (71.4%) at Glu-D1 loci. Three alleles, namely Glu-B1b (30.61%), Glu-B1c (25.85%) and Glu-B1i (34.00%) represented about 90% of the alleles at Glu-B1 locus. The combination of Glu-A1b, Glu-B1i and Glu-D1d alleles exhibited highest dough strength as measured by sedimentation value in comparison to other combinations (p<0.001). However, this combination was present only in 7% of the samples evaluated. In durum wheat, the null allele (Glu-A1c) was observed more frequently (76%) than the Glu-A1b allele (24%). Glu-B1f and Glu-B1e alleles represented equally (32% each). Protein subunits 13+16 and 6+8 were found correlated positively (p<0.05) with improved dough strength as compared to subunit 20 in durum wheat. This information can be a valuable reference for designing breeding programme for the improvement of bread and pasta making quality of bread and durum wheats, respectively in India.     

Analysis of diversity of Russian and Ukrainian bread wheat (Triticum aestivum L.) cultivars for high-molecular-weight glutenin subunits

The allelic diversity of high-moleculat-weght glutenin subunits (H WIGS) in Russian and Ukrainian bread wheat cultivars was analyzed. The diversity of spring wheat cultivars for alleles of the Glu-1 loci is characterized by medium values of the polymorphism index (polymorphism information content, PlC), and in winter wheats it varies from high at the Glu-A1 locus to low at the Glu-D1 locus. The spring and winter cultivars differ significantly in the frequencies of alleles of the glutenin loci. The combination of the Glu-A1b, Glu-B1c, and Glu-D1a alleles prevails among the spring cultivars, and the combination of the Glu-A1a, Glu-B1c, and Glu-D1d alleles prevails among the winter cultivars. The distribution of the Glu-1 alleles significantly depends on the moisture and heat supply in the region of origin of the cultivars. Drought resistance is associated with the Glu-D1a allele in the spring wheat and with the Glu-B1b allele in the winter wheat. The sources of the Glu-1 alleles were identified in the spring and wheat cultivars. The analysis of independence of the distribution of the spring and winter cultivars by the market classes and by the alleles of the HMWGS loci showed a highly significant association of the alleles of three Glu-1 loci with the market classes in foreign cultivars and independence or a weak association in the Russian and Ukrainian cultivars. This seems to be due to the absence of a statistically substantiated system of classification of the domestic cultivars on the basis of their quality.     

Changes in carbohydrates,amino acids and proteins in developing seed of chickpea

Developing seeds of chickpea cultivars G-130, L-550 and 850-3/27 grown under field conditions were sampled at different stages of maturity and analysed for soluble sugars, starch, soluble nitrogen, protein nitrogen and amino acids. Fr. wt of seeds of all three cultivars decreased after 28 days of flowering while the dry wt continued to increase. Rapid starch accumulation was observed between 14 and 28 days after flowering. Starch as per cent of seed dry wt started to decrease after 28 days, while starch per seed increased till maturity. The percentage of salt-soluble proteins decreased with maturation of seed. The electrophoretic pattern revealed that deposition of seed storage protein in cotyledons occurred 14 days after flowering. Most of the biochemical activity apparently occurred between 14 and 28 days after flowering.