摘要
偃麦草属是改良小麦产量和品质性状的重要基因资源。具有抗锈病、赤霉病和白粉病的多种抗性基因，亦具有大穗、多花、抗低磷营养胁迫、抗寒和抗旱等诸多优良性状。长穗偃麦草(Elytrigia elongata)是该属的一个二倍体物种，前人利用SDS-PAGE分析方法从中鉴定出高分子量谷蛋白亚基，通过培育染色体代换系的方法证实其编码基因位于1E染色体长臂。但有关偃麦草物种高分子量谷蛋基因分子序列的报道还非常少见。为认识该物种所具有的高分子量谷蛋白，对其基因编码的亚基进行了鉴定并克隆和测序了基因编码区。所获得的主要结果如下：
1．长穗偃麦草高分子量谷蛋白亚基组成分析
应用SDS-PAGE检测了8份二倍体长穗偃麦草的高分子量谷蛋白组成，检测结果表明均含有2-4个不等的亚基。选取其中1份材料P198526为重点研究对象，分别检测了33粒种子，发现有2-4条迁移率各异的高分子量谷蛋白亚基，但以3和4条的居多，显示该种子的高分子量谷蛋白亚基组成因共显而表现为杂合型。
2．长穗偃麦草y型Ee1.5和Ee1.8以及x型Ee1.9和Ee2.1高分子量谷蛋白基因编码区克隆及序列特点
以长穗偃麦草材料P198526单一植株DNA为模板，扩增获得4个分子量各别的高分子量谷蛋白亚基基因编码区片段，大小分别为1.5，1.8，1.9和2.1kb。克隆、测序和序列比较后分别命名为Ee1.5，Ee1.8，Ee1.9和Ee2.1。Genbank登录号分别为AY299518，AY298724，AY299520和AY299519。其中，Ee1.5，Ee1.8为y型，Ee1.9和Ee2.1为x型基因的编码区。
Ee1.5亚基全长编码区核苷酸长度为1492bp，可编码467个氨基酸残基的成熟蛋白。是目前明确的编码区最短的y型高分子量谷蛋白亚基之一，除具备y型高分子量谷蛋白亚基的典型特征外，其N保守端较其它同类亚基多出1个氨基酸残基，为105个。其序列结构与Dy10亚基十分类似，但其重复区中部有一额外的半胱氨酸，在所有y型亚基重复区末端都具备的一个半胱氨酸则消失了。
Ee1.8亚基全长编码区核苷酸长度为1788bp，可编码573个氨基酸残基的成熟蛋白。具备y型高分子量谷蛋白亚基的典型特征。
Ee1.9在其编码区内部存在一个提前终止密码子。不能编码可表达的成熟
蛋白。
Ee2.1亚基全长编码区核昔酸长度为2082bp，可编码670个氨基酸残基的
成熟蛋白。除具备x型高分子量谷蛋白亚基的典型特征外。在其重复区中部有
一额外的半肤氨酸，与Dxs重复区中额外半肤氨酸的部位不同。
由于所获得的来源于二倍体长穗堰麦草的高分子量谷蛋白基因编码区序列
与己报道的小麦同类基因在分子序列上存在较大差异，特别是N端保守区氨基
酸数目的增加，重复区内与二硫键形成至关重要的半肤氨酸的增加、减少以及
位置的改变将可能直接导致这些亚基在小麦面筋网络的形成过程中有不同寻常
的功能。因此，确有必要对这些亚基进行深入研究以期认识其在小麦品质改良
中的重要作用。
3.长穗僵麦草Ee染色体组高分子量谷蛋白基因与小麦同源基因的聚类分析
N端氨基酸序列聚类分析显示，Ee染色体组编码的y型高分子量谷蛋白因
Eel .8与Ay基因以较高的支持率聚类在一起。而Eel.5则与A、B和D染色体
组编码的亚基的分歧较大，以较高的支持率单独聚类为一枝。
而Ee染色体组编码的x型高分子量谷蛋白基因Eel .9(假基因)和Ee2.1与
小麦B染色体组编码的基因以较高的支持率聚类在一起。
Elytrigia possess many important genes or traits for wheat yield and quality improvement. Elytrigia elongata is one of the diploid species belonging to this genus. The HMW glutenin subunits and their structure gene locus on the long arms of IE chromosome (designated as Glu-E1) were confirmed by SDS-PAGE analysis of a set of Chinese spring (1A/1E, IB/IE, 1D/1E).E.elongata substitution lines. Except that, our knowledge about the HMW glutenin in this species was still very limited. In order to get better understanding the HMW glutenin in E. elongata, the HMW glutenin subunits compositions and their coding sequences were analysis and sequenced. The main results were as follows:
1. HMW glutenin subunit compositions in Elytrigia elongata
Two to four HMW glutenin subunits could be detected in each of the 8 accessions of E. elongata by SDS-PAGE analysis. To clear the reason why up to four HMW glutenin subunits can be detected in this diploid species, thirty-three different seeds of one accession PI 98526 were further analyzed. Similarly, two to four HMW subunits could still be detected and every grain was different from each other in HMW glutenin subunit compositions. Interestingly, three or four HMW glutenin subunits appeared with the highest frequency of all, indicating that the seeds were heterogeneous and the HMW glutenin were co-expressesed.
2. Molecular cloning and sequences analysis of HMW glutenin subunits Ee1.5, Ee1.8, Ee1.9 and Ee2.1
Four PCR fragments of the putative HMW glutenin coding regions, ranging from approximately 1.5 to 2.1 kb, were obtained using the genomic DNA from the single plant of PI98526. Four types of positive clones were obtained and selected to represent the complete coding regions of the 1Ex and lEy subunits, respectively. Using a set of subclones prepared by nested deletion, the four HMW glutenin coding sequences, pEel.5, pEel.8, pEel.9 and pEe2.1, were completely sequenced. They have been deposited in the Genbank under accession numbers AY299518, AY298724, AY299520 (pseudogene) and AY299519, respectively.
The coding sequence of y type glutenin subunit Ee1.5 had 1492bp in nucleotide and was one of HMW glutenin subunits with the shortest coding regions. In contrast with other y-type HMW glutenin subunits, the N-terminal domain of Eel.5 had 105 but not 104 amino acid residues. Comparison of Eel.5 with Dy10 suggested that Ee1.5 had an extra Cys residue at the middle of repetitive domain, while; the typical
Cys residue at the end of repetitive domain in all other y-type HMW glutenin was lost. The coding sequence of another typical y-type HMW glutenin Ee1.8 was made up by 1788bp in nucleotide and could encode a mature protein of 573 amino acid residues.Ee1.9 was a pseudogene and couldn' t encode any mature protein because of an inframe stop code. Ee2.1 was a typical x type HMW glutenin subunit. The coding sequence of Ee2.1 was made up by 2082bp in nucleotide and could encode a mature protein of 670 amino acid residues. An extra Cys residue was found at the central repetitive domain: Another x-type HMW glutenin with an extra Cys residue in repetitive domain was DX5. While, the extra Cys in Dx5 was not at central but at the beginning of repetitive domain, which made Dx5 had better quality score than Dx2 without an extra Cys in this position.
It was clear that the coding sequences of HMW glutenin isolated from Elytrigia elongate have novel modification. Such as, an extra amino acid residue was founded in N-terminal and extra Cys was found or lost in repetitive domain and changed in position. All of these changes would endow these HMW glutenin have the potentially better quality scores than those of studied before. So, it was necessary for further understanding the quality functional of these glutenin isolated from E. elongate.
3. Phylogenetic relationships of HMW glutenin subunits encoded by A, B and D genome of wheat and by Ee genome of Elytrigia elongata
In the phylogenetic tree made up by N-terminal residues, two x-type subunits Ee1.9 and Ee2.1 were clustered together with Bx7 in high
引文
1 Dong Y-C(董玉琛),Zheng D-S(郑殿升). Genetic resource of Chinese wheat. Pekin: China agricultural publishing company, 1998
2 Wan, Y F, C. Yen, Y L Zheng. Sources of resistance to head scab in Triticum. Euphytica, 1997, 94:321～323
3 Wang C-Y(王长有), Ji W-Q(吉万全),Xue X-Z(薛秀庄),et al. Studies on yellow rustresistance of T. aestivum Th. intermedium alien disomic addition iines.Acta Bot. Boreal. -Occident. Sin<西北植物学报>,1999,19(6):54～58(in Chinese with English abstract)
4 马渐新，周荣华，董玉琛，等．来自长穗偃麦草的抗小麦条锈病基因的定位．科学通报，1999，44(1)：65～69
5 张增艳，辛志勇，马有志，等．用分子标记定位源于中间偃麦草的小麦抗黄矮病基因．中国科学(C辑)，1999，29(4)：413～417
6 Li Y-J(李玉京)，LIu J-Z(刘建中)，Li B(李滨)，et al．Chromosomal Location of the Genes
Conferring the Tolerance to Phosphorus Deficiency Stress in Lophopyrum elongatum Genome. Acta Genetica Sinica,<遗传学报> 1999,26(6):703～710(in Chinese with English abstract)
7 Zhang R-Q(张荣琦),Chen C-H(陈春环),Zhao X-N(赵晓农),et al.A study on the development with drought resistance of wheat variety through crossesbetween octoploid tritielytrigia typesand triticum aestivum. Acta Bot.Boreal.-Occident. Sin<西北植物学报 >,1999,19(1):24～28(in Chinese with English abstract)
8 Lawrence G J, Shepherd. Chromosomal location of genes controlling seed proteins in species related to wheat. Theor Appl Genet, 1981,59:25～31
9 Dvorak J, Kasarda D D, Dietler M D, et al. Chromosomal location of seed storage protein genes in the genome of Elytrugia elongata. Can.J.Genet.Cytol, 1986,28:818～830
10 Zhang Y-B(张延滨),Qi S-Y(祁适雨),Xiao Z-M(肖志敏),et al. Genetic analysis of biochemical finger print sofoctoploid of wheat-wheatgrass(ZHONGl-ZHONG7) and Thinopyrum intermedium by SDS-PAGE and A-PAGE. NATURAL SCIENCE JOURNAL OF HARBINNORMAL UNIVERSITY<哈尔滨师范大学自然科学学报>, 1998,14(1): 84～90
11 Fan S-H(范三红),Guo A-G(郭蔼光).A study on the origin of HMW-GS1 4and1 5in Xiaoyan 6.Acta Univ.Agric. Boreali- occidentalis<西北农业大学学报>,2000,28(6):1～5(in Chinese with English abstract)
12 Yan Z-H(颜泽洪), zheng Y-L(郑有良), Wan Y-F(万永芳), et al.粗山羊草高分子量麦谷蛋白新型亚基的筛选和鉴定.四川农业大学学报, 2001, 19(3): 197～199
13 Wan Y, Wang D, Shewry P R, et al. Isolation and Characterization of five novel high molecular weight subunit of glutenin genes from Triticum timopheevi and Aegilops cylindrical. Theor Appl Genet,2002, 104:828～839
14 Yan Z, Wan Y, Liu K, et al. Identification of a novel HMW glutenin subunit and comparison of its amino acid sequence with those of homologous subunits. Chinese Science Bulletin,2002,47(3):220～225
15 Tang Z-H(唐朝晖), Liu S-B(刘守斌), You M-S(尤明山), et al. Expression, Chromosomal Location and Molecular Marker of High-Molecular-Weight Glutenin SUbunit Gene of Thinopyrum elongatum in Wheat Background. Journal of Agriculture Biotechnology < 农业生物技术学报>,2002,11(1):34～39(in Chinese with English abstract)