山羊草物种高分子量麦谷蛋白基因的分子克隆

简介：作者 颜泽洪，男，1970年08月出生，于2001年07月获博士学位。2004年度全国优秀博士学位论文，导师 郑有良教授

高分子量麦谷蛋白是成熟小麦种子中的主要贮藏蛋白之一，其组成和数量可直接影响普通小麦的品质。而在栽培小麦中所发现的优质高分子量麦谷蛋白亚基的类型并不多。山羊草属(Aegilops）是小麦的近缘属之一，山羊草物种中的高分子量麦谷蛋白亚基的遗传变异类型较普通小麦基因组编码的更丰富，为了进一步深入了解这些物种所具有的高分子量麦谷蛋白亚基与普通小麦中所发现的亚基在分子水平的差异，以及将它们用于改良普通小麦品质的应用前景，对粗山羊草和尾状山羊草的高分子量麦谷蛋白亚基进行了分析、鉴定和分子克隆。所获得的主要结果如下：

1．粗山羊草高分子量麦谷蛋白亚基的组成分析

利用SDS-PAGE和Western-Blotting 分析与鉴定技术，从51份山羊草中共发现了2种新型亚基Tx>2和Ty<12 , 被命名为dx2.1t和dy13t 和7种亚基组合类型(2+12、5+10、2+10、5+12、2.1t +10、2.1t +12和2+13 t。这7种亚基组合的频率分布是27.5%、25.5%、31.4%、3.9%、2.0%、2.0%和7.8%。

2．粗山羊草Dy13t亚基的分子克隆

粗山羊草的Dy13t亚基的全长编码区有1878个核苷酸。由其所推导的氨基酸序列包括一个含有21个氨基酸残基的信号肽, 一个含有104个氨基酸残基的N-端保守区,一个含42个氨基酸残基的C-末端保守区，以及一个含有457个氨基酸残基的重复区。重复区中含有47个六肽(PGQGQQ)，17个九肽(GYYPTSLQQ)，以及位于重复区起始端的十二肽(GYYPSVTSPRQG)和十一肽(SYYPGQASPQQ)和重复区末端的三肽(GYD)。该亚基是目前所知的由D基因组编码的y 型高分子量麦谷蛋白亚基中重复区最短的。它与普通小麦的Dy10和Dy12有较大的差异，因而是1种新型的高分子量麦谷蛋白亚基，对普通小麦的品质改良可能有重要的利用价值。

从3个具有一致迁移率的粗山羊草材料As2395，As2396和As2386中，所获得的亚基在核苷酸和氨基酸水平的差异是极小的，虽然它们或来源于不同地方(As2395，As2396与As2386),或分属不同的亚种Aegilops tauschii, ssp.eusquarrosa (As2395), Aegilops tauschii, ssp.strangulata (As2396和As2386)，但其高分子量麦谷蛋白亚基的差异是很小的。

3．粗山羊草Dx2.1t亚基的分子克隆

粗山羊草的Dx2.1t亚基的全长编码区含有2514个核苷酸。由其所推导的氨基酸序列包括一个含有21个氨基酸残基的信号肽，一个含有89个氨基酸残基的N-端保守区，一个含42个氨基酸残基的C-末端保守区以及一个含有684个氨基酸残基的重复区。重复区中含有71个六肽(PGQGQQ)，19个九肽(GYYPTSLQQ)，20个三肽(GQQ)，以及位于重复区起始端的十二肽(RYYPSVTSPQQV)和十一肽 (SYYPGQASPQR)和重复区末端的三肽(GYY)。该亚基是一种不同于普通小麦Dx2和Dx5的新型亚基，它对普通小麦的品质改良的利用价值值得进一步深入研究。

4．尾状山羊草Cx亚基的分子克隆

尾状山羊草Cx亚基的全长编码区含2391个核苷酸。由其所推导的氨基酸序列包括一个含有21个氨基酸残基的信号肽, 一个含有86个氨基酸残基的N-端保守区,一个含42个氨基酸残基的C-末端保守区以及一个含有646个氨基酸残基的重复区。重复区中含有66个六肽(PGQGQQ),18个九肽(GYYPTSLQQ)和21个三肽(GQQ),以及位于重复区起始端的十二肽(RYYPSVTSPQQV)和十一肽(SYYPGQASPQR)和重复区末端的三肽(GYY)。该亚基不同于小麦族A、B、D、R染色体组编码的高分子量麦谷蛋白亚基，是小麦族物种高分子量麦谷蛋白亚基大家族的新成员，它在系统进化上与普通小麦D基因组编码的高分子量麦谷蛋白亚基的亲源关系最近，因而是值得进一步研究利用的新型亚基。

5. 尾状山羊草Cy亚基的分子克隆

尾状山羊草Cy亚基的全长编码区含1905个核苷酸。由其所推导的氨基酸序列包括一个含有21个氨基酸残基的信号肽, 一个含有104个氨基酸残基的N-端保守区,一个含42个氨基酸残基的C-末端保守区以及一个含有464个氨基酸残基的重复区。重复区中含有50个六肽(PGQGQQ),16个九肽(GYYPTSLQQ),以及位于重复区起始端的十二肽(GYYPSVTSPHQS)和十一肽(SYYPGQASPQR)和重复区末端的三肽(GYD)。该亚基与小麦族A、B、D、R染色体组编码的高分子量麦谷蛋白亚基的差异较大，是小麦族物种高分子量麦谷蛋白亚基大家族的新成员。

6.所克隆高分子麦谷蛋白基因的聚类分析

Dy13t在系统进化树中与D基因组编码的y亚基聚类在一起。虽然Dy10, Dy12, Dy12t和Dy13t均来自D基因组，但来自普通小麦D基因组的Dy10和Dy12亚基间和粗山羊草D基因组的Dy12t和Dy13t亚基间的亲缘关系更近。Dy13t亚基与A、B和R染色体组编码的y亚基的亲缘关系相对较远。 Dx2.1t则与Dx2和Dx5的亲缘关系更近。与A、B和R染色体组编码的x亚基的亲缘关系较远。Cx亚基与D基因组编码的亚基的亲源关系最近，与R基因组编码的亚基的亲源关系略远，再其次是A基因组编码的亚基,与B基因组编码的亚基亲源关系最远。来自二倍体尾状山羊草(CC)的Cy亚基与四倍体的柱穗山羊草(CCDD)的Cy亚基的亲源关系最近，与A和R基因组的亚基的亲源关系较近，与B和D基因组编码的亚基的亲源关系较远。

7．所克隆高分子量麦谷蛋白基因的体外表达

Dx2.1t、Cx、Cy 3个基因均在细菌中获得表达，经过SDS-PAGE 分析和Western-Blotting 鉴定表明所表达的蛋白与种子中的相应的高分子量麦谷蛋白完全一致。可以大量提纯所表达的高分子量麦谷蛋白亚基将其添加到普通小麦面粉中以研究其对普通小麦品质改良的贡献。

关键词：小麦(Triticum aestivum)、山羊草属(Aegilops)、粗山羊草(Aegilops tauschii)、尾状山羊草(Aegilops caudata)、高分子量麦谷蛋白亚基、分子克隆、 体外表达

Abstract

High molecular weight (HMW) glutenin subunits, as one of the seed storage proteins in wheat, its composition and amount have a profound influence on the baking quality of wheat. The number of functionally superior HMW glutenin subunits identified in common wheat is very limited. The Aegilops genusis very closely related to the common wheat. The types of HMW glutenin subunits in Aegilops species are much richer than those in bread wheat. For the better understanding the molecular structure difference of these HMW glutenin subunits specified by the Aegilops species and those of wheat and their potentially utilization for the wheat quality improvement,the HMW glutenin subunits composition of 51 accessions from Aegilops tauschii(DD,2n=2x=14) and 1 accession from Aegilops caudata(CC,2n=2X=14) were studied using SDS-PAGE and Western-blotting. In addition, Six novel HMW glutenin genes were isolated and expressed in E.coli.

1.HMW glutenin subunit composition analysis of Aegilops tauschii

Two novel types of HMW glutenin subunits were observed from 51 accessions of Aegilops tauschii ,which were designated as Dx2.1t and Dy13t,respectively.The frequencies of 7 subunit combinations(2+12,5+10,2+10,5+12,2.1t+10,2.1t+12 and 2+13t) are 27.5%(2+12),25.5%(5+10) ,31.4%(2+10),3.9%(5+12),2.0% (2.1t+10), 2.0%(2.1t+12)and 7.8%(2+13t),respectively

2.Molecular cloning of Dy13t subunit gene from Aegilops tauschii

The complete nucleotide sequence of the Dy13t subunit contained 1878 base pairs. The deduced amino acid sequence of Dy13t contained a signal peptide of 21 amino acids, a N-terminal region of 104 amino acids, a C-terminal region of 42 amino acids and a central repetitive domain of 457 amino acids. There were 47 hexapeptides (PGQGQQ), 17 nonapeptides (GYYPTSLQQ), two peptides containing, respectively, 12(GYYPSVTSPRQG) and 11(SYYPGQASPQQ) and a tripeptide (GYD) in the repetitive domain. Among all the known HMW glutenin subunits encoded by D-genome, the coding region for Dy13t was the shortest. Its sequences is obviously difference from Dy10 and Dy12. Therefore , Dy13t is a novel subunit characterized and has an important utilization in the wheat quality improvement.

3.Molecular cloning of Dx2.1t subunit from Aegilops tauschii

The complete nucleotide sequence of the Dx2.1t subunit contained 2514 base pairs. The deduced amino acid sequence of Dx2.1t contained a signal peptide of 21 amino acids, a N-terminal region of 89 amino acids, a C-terminal region of 42 amino acids and a central repetitive domain of 684 amino acids. Exception for hexaploid (PGQGQQ) and nonapeptides (GYYPTSLQQ), many additions tripeptides(GQQ) were detected in the repetitive domain

4.Molecular cloning of Cx and cy subunit from Aegilops Caudata

The complete nucleotide sequence of the Cx and Cy genes contained 2391 and 1905 base pairs. The proteins encoded by these two genes consisted of signal peptide, a non-repetitive N-terminal and a C-terminal region, flanked by a repetitive domain.The great differences in amino acid composition and order of hexpeptides and nonapeptides between Cx and Cy subunits and other HMW subunits in bread wheat indicated that they are new members of HMW glutenin family.

5 . Phylogenetic relationships of Dy13t,Dx2.1t,Cx and Cy

In the Phylogenetic tree map,Dy13t is very closely related to the y-type genes encoded by D genome of common wheat. Although Dy10,Dy12,Dy12t,and Dy13t are all encoded by the D genome ,the Dy13t is more closed related to the Dy12t than both Dy10 and Dy12. Dx2.1t is more closely clustered to both Dx2 and Dx5. The Cx is closed associated with D genome subunits compared with R and A genome, while Cy is very closely related to the Cy from Aegilops cylindrica(2n=4x=28,CCDD).

6. Expression of High molecular weight glutenin subunits in E.coli

Three high molecular weight glutenin subunits(Dx2.1t,Cx,Cy)have been successfully expressed in E.coli (BL21(DE3)plys). The expressed HMW glutenin subunits of cloned genes in E.coli have the same mobilities to those of seeds in SDS-PAGE,They can be detected by the polyclonal antibody recognizing HMW glutenin subunit . The high level of expression of cloned HMW glutenin genes plays an important role in further study on function of protein and effects on wheat dough by incorporation into wheat flour.

KeyWords: Wheat(Triticum aestivum) , Aegilops tauschii, Aegilops caudate, High molecular weight glutenin subunit, Molecular cloning, Protein expression