【作者】 赵继新；

【导师】 吉万全； 陈新宏；

【作者基本信息】 西北农林科技大学 ， 作物遗传育种， 2010， 博士

【摘要】 华山新麦草(Psathyrostachys huashanica Keng.)是我国一个特有种,为国家一类珍稀濒危植物和急需保护的农作物野生亲缘物种;具有抗寒、抗旱、耐瘠薄、早熟、优质、矮杆、抗病等特点。本研究通过细胞学、原位杂交和PAGE电泳鉴定出携带华山新麦草的HMW-GS、LMW-GS和醇溶蛋白基因的华山新麦草1Ns二体异附加系,并对该异附加系进行了农艺性状、品质特性和SSR标记研究,同时对华山新麦草的HMW-GS、LMW-GS和醇溶蛋白基因进行了克隆和序列分析。主要结果如下:1、在小麦与华山新麦草的杂交和回交后代中,选育出小麦-华山新麦草二体异附加系H9021-28-5。对其进行细胞遗传学研究表明:H9021-28-5的染色体数目及构型为2n=44=22Ⅱ,遗传基本稳定;荧光原位杂交表明,H9021-28-5包含42条小麦染色体和1对华山新麦草染色体;种子贮藏蛋白聚丙烯酰胺凝胶电泳(SDS-PAGE和A-PAGE)表明:H9021-28-5携带有华山新麦草的高分子量谷蛋白亚基(HMW-GS)、低分子量谷蛋白亚基(LMW-GS)和醇溶蛋白(Gliadin)基因。说明,H9021-28-5为普通小麦-华山新麦草的1Ns二体异附加系,且华山新麦草的HMW-GS、LMW-GS和醇溶蛋白(Gliadin)基因已导入普通小麦。2、对异附加系H9021-28-5的农艺性状调查表明,附加华山新麦草1Ns染色体后,小麦株高、穗长、小穗数、穗粒数等性状显著降低,小麦籽粒的长度和粒径等性状显著升高,条锈病抗性增强。面粉加工品质分析表明,含有华山新麦草HMW-GS、LMW-GS和醇溶蛋白基因的异附加系比小麦亲本7182,在沉降值、蛋白质含量、面团形成时间、稳定时间、拉伸曲线面积、拉伸阻力和延伸度等品质指标上得到明显提高。矿物质元素含量测定表明,华山新麦草的多种矿物质元素含量均比普通小麦高,异附加系H9021-28-5的钙、磷、镁、锌等11种元素的含量得到明显提高。SSR和EST-SSR标记分析表明,在278对SSR引物中有Xgdm19、Xgdm43、Xgwm213和Xgwm497等4对引物,在49对EST-SSR引物中有Ksum11、Ksum19、Ksum117、Ksum220、Ksum255和Swes145共6对引物,在华山新麦草和异附加系H9021-28-5中均可扩增出特异条带,这些引物可初步用来检测华山新麦草的1Ns染色体。3、采用PCR方法,从华山新麦草基因组中分离到了华山新麦草HMW-GS的启动子基因序列。对该启动子基因HGp-Ns-1(GQ139524)序列分析显示,该序列在起始位点上游,从5’至3’侧翼方向,依次有E-box,N-box,G-box,HMW谷蛋白特异38bp增强子序列和TATA-box等HMW-GS特异表达的顺式或反式作用调控元件;系统进化分析表明,该启动子序列(HGp-Ns-1)与拟鹅观草Ps. spicata的y-型HMW-GS启动子基因具有相对较近的亲缘关系。4、通过PCR方法,从华山新麦草基因组中分离克隆到了4条LMW-GS基因序列。序列结构分析显示,这4条序列的编码区依次都有信号肽、N-末端保守区、中间重复区、C-末端I区、C-末端II区和C-末端保守区等特征区域,都含有8个半胱氨酸残基(C),N-末端起始氨基酸序列为METSRIPS-或METSRIPG-,说明,这4条序列都为华山新麦草的LMW-m型LMW-GS基因。序列FJ600493(LG-Ns-1)、FJ600494(LG-Ns-2)和GQ223386(LG-Ns-3)在编码区出现提前终止密码子,可能为假基因。序列GQ139528(LG-Ns-4)包含LMW-GS的基因完整编码区和部分启动子序列,该启动子序列的5’侧翼端,从5’到3’方向,依次有保守胚乳框、E-box、F-box、CAAT box、TATA框等LMW-GS基因的特异表达调控元件。这4条序列的系统进化分析表明,它们与纤毛鹅观草E. ciliaris的LMW-GS基因具有相对较近的亲缘关系。5、采用PCR方法,从华山新麦草基因组中分离获得了4条α-型和1条ω-型醇溶蛋白基因序列。对4条α-醇溶蛋白基因序列结构分析表明,这4条序列都依次含有信号肽、N-末端重复区、多聚谷氨酰胺Ⅰ区、特征区Ⅰ、多聚谷氨酰胺Ⅱ区和特征区Ⅱ等结构区域;序列Gli-Ns-2(FJ713595)在特征区Ⅰ出现了2个提前终止密码子,可能为假基因。序列Gli-Ns-2和Gli-Ns-3(GQ139525)的重复单元为P(F/Y)P(P/Q)(P/Q)Q;这4条序列都含有8或9个半胱氨酸残基;系统进化分析表明,序列FJ713595与柔软赖草和纤毛鹅观草的α-醇溶蛋白基因具有相对较近的亲缘关系,序列Gli-Ns-3、Gli-Ns-4(GQ139526)和Gli-Ns-5(GQ139527)可能为α-醇溶蛋白基因家族中的新类型。对获得的1条ω-醇溶蛋白基因Gli-Ns-1(FJ600500)序列结构分析表明,该序列具有ω-醇溶蛋白典型的信号肽、N-末端区和部分重复区序列,但在重复区内出现了1个提前终止密码子,可能为假基因。系统进化分析显示,其可能为ω-醇溶蛋白基因家族中的新类型。华山新麦草的HMW-GS、LMW-GS和醇溶蛋白基因的分子克隆和序列分析,以及华山新麦草1Ns异附加系的培育及其品质效应的研究,对丰富小麦品质遗传种质资源、研究和利用华山新麦草优异基因、开发和保护这一珍稀濒危物种具有重要意义。更多还原

【Abstract】 Psathyrostachys huashanica Keng. (2n = 2x = 14, NsNs) is an endemic species in China that has been listed as an endangered and imperatively protected wild species. It is characterized by early maturity and resistance to drought, salinity, wheat take-all fungus and stripe rust. In this paper, a wheat-P. huashanica 1Ns disomic addition line“H9021-28-5”was developed and its chromosomal configuration and behaviors were analyzed, and the high molecular weight glutenin subunit (HMW-GS), low molecular weight glutenin subunit (LMW-GS) and gliadin genes of P. huashanica were identified. The agronomic traits, the quality effect, and the mineral element content of this alien addition line were surveyed. The HMW-GS, LMW-GS and gliadin genes of P. huashanica were molecular cloned and the sequence were analyzed. Main results are as follows:1) The Cytogenetics investigations revealed that the chromosome number and configuration of“H9021-28-5”were 2n = 44 = 22 II. The mitotic and meiotic GISH analysis indicated that“H9021-28-5”contained 42 wheat chromosomes and a pair of P. huashanica chromosomes. The SDS-PAGE and A-PAGE analysis showed that“H9021-28-5”carried the HMW-GS, LMW-GS and gliadin genes of P. huashanica. The results suggest that these storage protein genes of P. huashanica had been transferred into common wheat, and“H9021-28-5”is a wheat-P. huashanica 1Ns disomic addition line.2) The agronomic traits survey on H9021-28-5 indicated that P. huashanica 1Ns chromosome may be have a reduced effect on the wheat plant height, length of spike, No. of spikelets, and grains per spike, and may be have a stimulating effect on grain weight, length, diameter, and hardness. The processing quality analysis indicated that H9021-28-5 have been significantly improved in the sedimentation value, protein content, dough development time, stability time, extention resistance, and dough extensibility. Determination of mineral elements shows that many kinds of element content in P. huashanica is higher than wheat, and 11 kinds of element content in alien addition line H9021-28-5 have been markedly enhanced, compared with wheat cv. 7182.. The SSR and EST-SSR markers analysis showed that, there are four pairs of SSR primers and six pairs of EST-SSR primers can be preliminary used as P. huashanica 1Ns chromosome-specific molecular markers. 3) The HMW-GS promoter of P. huashanica were cloned by PCR method, and its sequences were analyzed. Analysis on this promoter gene HGp-Ns-1 (GQ139524) showed that the sequence contain an E-box, N-box, G-box, HMW-endosperm-specific 38bp enhancer and TATA-box. Phylogenetic analysis indicated that HGp-Ns-1 has a relatively closer relationhip with the y-type HMW-GS promoter gene of Ps. spicata.4) The four LMW-GS gene of P. huashanica were isolated, and sequences analysis showed that these four sequences all included signal peptide, N-terminal region, the repetitive domain, and C-terminal region, successively. There are eight cysteine residues in coding region. Starting as METSRIPSL- or METSRIPGL- in N-terminal domain indicated that these 4 sequences are the m-type LMW-GS gene. FJ600493 (LG-Ns-1), FJ600494 (LG-Ns-2) and GQ223386 (LG-Ns-3) may be pseudogene because of two premature stop codons appeared in coding sequences. Structural analysis on GQ139528 (LG-Ns-3) revealed that it contained a part of LMW-GS promoter gene and coding region of LMW-GS. Promoter sequence analysis showed that there have the conservative endosperm box, E-box, F-box, CAAT box, and TATA box. Phylogenetic analysis suggested that these four P. huashanica LMW-GS genes have a relatively closer relationship with the E. ciliaris LMW-GS genes.5) The fourα-type and aω-type gliadin gene sequences were isolated from P. huashanica genomes. Sequences structural analysis on fourα-gliadins revealed that these sequences contain signal peptide, N-terminal repetitive domain, polyglutamine domain I, unique domain I, polyglutamine domain II, and unique II, successively. Sequence Gli-Ns-2 (FJ713595) may be a pseudogene because of two premature stop codons appeared in unique domain I. There 8 or 9 cysteine residues in the four sequences. Sequence structure analysis on aω-gliadin gene Gli-Ns-1 (FJ600500) showed that it have signal peptide, N-terminal region and part of repetitive domain of typical structure ofω-gliadin, and it may be a pseudogene because of a premature stop codons appeared in repetitive domain. Phylogenetic analysis suggested that FJ713595 have closer kinship withα-gliadin genes of leymus mollis and E. ciliaris. Sequence Gli-Ns-4 (GQ139525), Gli-Ns-4 (GQ139526), Gli-Ns-5 (GQ139527), and FJ600500 may be a new type of prolamin gene family.These reseach are of important significance for enriching and improving wheat resources of storage proteins genes, and for continuing to exploit the advantageous genes of the endangered species P. huashanica.更多还原