【作者】 杨在君；

【导师】 彭正松； 周永红；

【作者基本信息】 四川农业大学 ， 植物资源保护与利用， 2011， 博士

【摘要】 小麦是世界上最重要的粮食作物之一。随着人口的剧增,可耕种土地的减少,目前世界小麦的产量已呈逐年下滑的趋势,因此当前小麦育种的主要目标仍然是增加小麦产量。增加小麦穗粒数是增加产量的一条有效途径,而小麦穗粒数是小麦花分化、发育、退化与结实等一系列生理过程的最终体现。小麦三雌蕊突变体(TP)是由彭正松先生培育出的一新的突变体,TP是由一对显性核基因控制,与细胞质遗传无关。三雌蕊突变体与Murai等发现的雄蕊雌蕊化品系不同,三雌蕊突变体具有3个正常的雄蕊和3个可育的雌蕊,一朵小花可结三粒种子,能显著的增加穗粒数,因而具有一定的育种学价值。利用微卫星(SSR)分析已将控制三雌蕊性状的Pis1基因定位于2D染色体的长臂上。小麦三雌蕊突变体是研究小麦雌蕊发育的理想材料,虽然目前小麦三雌蕊突变体的遗传基础基本清楚,但控制三雌蕊发生的Pis1基因的DNA序列信息尚不清楚。三雌蕊性状的发生是受一显性单基因控制,但该基因会引发一系列基因的表达差异。因此可以肯定的是三雌蕊性状的形成是一系列基因的差异表达的结果,因此本研究的目的是分离小麦三雌蕊突变体在幼穗发育过程中差异表达的基因。主要研究结果如下：1.以三雌蕊突变体(TP)为供体,单雌蕊地方品种中国春(CS)、川麦28(CM28)、绵阳29(MY29)、内麦9号(NM9)为轮回亲本,经7代回交,4代自交,初步培育出三雌蕊近等基因系CSTP, CM28TP, MY29TP, NM9TP。利用128对SRAP引物对培育的近等基因系及轮回亲本进行SRAP分析,结果表明：(1)128对引物共扩增出978个条带。其中有120对引物的扩增产物具有多态性,占所用引物的93.8%。这120对引物共扩增出638个差异条带,占总条带数的65.2%;(2)利用128对SRAP引物计算9个材料之间的遗传相似系数。其中CS与CSTP的相似系数为0.9346,MY29与MY29TP的遗传相似系数为0.9070,CM28与CM28TP的遗传相似系数为0.9397,NM9与NM9TP的遗传相似系数为0.8732。(3)通过聚类分析筛选出两对遗传相似性大于0.93的近等基因系,即CM28TP与CM28、CSTP与CS。2.利用两对三雌蕊性状的近等基因系CM28TP和CSTP以及它们的轮回亲本构建了小麦三雌蕊性状的正向抑制性差减杂交cDNA文库。从CM28TP cDNA文库中分离出62个差异表达基因,其中30个基因为已知基因,它们编码的蛋白质主要用于调控蛋白合成、信号转导以及离子运输等。其余32个基因为未知基因。从CSTP cDNA文库中分离出161个差异表达基因,其中67%的基因为未知基因,剩下的33%的基因为已知基因。通过序列比对发现有四个基因,即：硫氧还蛋白H,泛素连接酶,微小染色体维持蛋白2(MCM2),和泛醇细胞色素C还原酶复合体14kD蛋白在两个近等基因系CM28TP和CSTP中均过量表达。虽然CM28TP和CSTP具有不同的遗传背景,但它们都表现为三雌蕊性状,因此推测这四个基因与三雌蕊的发育相关。3.利用引物退火控制技术对小麦三雌蕊性状的两对近等基因系CM28TP和CSTP及其轮回亲本CM28和CS幼穗中差异表达基因进行分离。利用120条ACP引物,我们从CM28TP和CSTP中分离出3个差异表达基因,并将其分别命名为DETP-1, DETP-2和DETP-3。通过Blastx比对分析发现,DETP-1与玉米中的细胞质膜蛋白基因同源,而细胞质膜蛋白在细菌中的功能是调控细胞分化。DETP-3与玉米的内切-1,4-β-葡聚糖酶(EGases)基因同源。EGases基因在植物的发育、细胞壁的收缩、花轴及根的延长方面起作重要的作用。DETP-2在Genebank中没有找到与之同源的基因,可能是一新的基因。通过Real-time PCR分析,DETP-1, DETP-2和DETP-3在CM28TP和CSTP中的表达量均高于其轮回亲本CM28和CS,因此推测这三个基因可能与三雌蕊的发育有关。4.利用RT-PCR技术从小麦三雌蕊突变体(TP)中克隆出一个RING finger泛素蛋白连接酶基因的全长cDNA,我们将其命名为TaZFP-1.对该cDNA核苷酸序列、氨基酸序列、理化性质、疏水/亲水性、二级结构、结构域、与其它植物的RING finger泛素蛋白连接酶的同源比较和系统进化树进行了预测和分析。结果表明：TaZFP-1基因共759个核苷酸编码252个氨基酸,蛋白质的分子量为27.9 kD,理论等电点PI为6.23；TaZFP-1蛋白的大部分氨基酸为亲水性氨基酸,因此推测TaZFP-1是一可溶性蛋白；二级结构预测表明,TaZFP-1蛋白以α螺旋、β折叠和无规则卷曲为主；通过结构域分析发现,TaZFP-1蛋白在196～233位氨基酸之间有一保守的结构域,该结构域中富含半胱氨酸,形成典型的C3HC4锌指结构；同源序列比对发现不同植物的RING finger泛素蛋白连接酶的氨基酸序列差异较大,但在C端都具有C3HC4锌指结构的保守区；进化树分析表明TaZFP-1蛋白与水稻的RING finger泛素蛋白连接酶的相似性最高,为73%。推测TaZFP-1与水稻的RING finger泛素蛋白连接酶具有相似的功能。5.利用RT-PCR与RACE相结合的方法,我们从小麦三雌蕊突变体(TP)幼穗中克隆出一内切-1,4-β-葡聚糖酶(EGases)基因的全长cDNA序列,并将其命名为TaEG。对该序列的核苷酸序列、氨基酸序列、.理化性质、疏水/亲水性、二级结构和三级结构、跨膜结构域以及与其他植物EGases的同源性进行了预测和分析。结构表明：TaEG的全长cDNA序列由2174个核苷酸组成,编码622个氨基酸,蛋白质的分子量为69.07kD,理论等电点PI为9.39；TaEG的大部分氨基酸序列为亲水性氨基酸,因此推测TaEG蛋白为一可溶性蛋白；二级结构预测表明,TaEG蛋白以α螺旋、β折叠和无规则卷曲为主；跨膜结构分析发现,在TaGE蛋白的N端74～96位氨基酸处形成一跨膜区域,因此推测TaGE是一跨膜蛋白型EGase。聚类分析.结果同样证实TaGE是一跨膜蛋白型EGase。本研究为深入探讨TaGE的性质及其在小麦雌蕊发育中的作用奠定了基础。更多还原

【Abstract】 Wheat(Triticum aestivum L.) is the most important food crop in the world and becomes even more important as the global population increases. Because the arable land is very limited, improving the yield is essential. One way to improve wheat yield potential is to increase grain number per spike. The grain number of per spike is the ultimate embodiment of the floral differentiation, degeneration and grain-setting. The common wheat line three pistils (TP), which selected by Peng from the’trigrain’ wheat variety, is a valuable mutant for the wheat breeding. Because the TP mutation has normal spike morphology but produces 3 pistils per floret, it has potential to increase grain number per spike. Homeotic transformation of stamens into pistil-like structures (pistillody) is caused by nuclear-cytoplasm interaction in wheat. The TP mutation is different from the pistillody, because the 3 stamens and 3 pistils are all fertile. Genetic analysis indicated that the three pistils trait is controlled by a single dominant gene Pis1 which was located on chromosome 2DL. Up to now, the genetic basis of the three pistils trait has been clearly, but the sequence information of Pis1 is still unclear. Although the three pistils controlled by a single gene locus, the mutation causes changes of a series of genes at the transcription level and affect various metabolism processes in three pistils plants. So the aim of this study was to identify differentially expressed genes in young spikes of three pistils plants, in order to reveal the molecular mechanism controlling the three pistils characters. The main results are summarized as follows:1. In this study, three pistils (TP) was used as donor parent, and Chinese spring (CS), Chuanmai 28 (CM28), Mianyang 29 (MY29) and Neimai 9 (NM9) were used as recurrent male parent, after 7 times successive backcrossing and then self-crossing for four generations, four near isogentic lines (NILs), CSTP, CM28TP, MY29TP and NM9TP were bred. Genetic similarity and genetic distance of 4 near isogentic lines and their recurrent parents were compared and analysised by SRAP. The results showed:(1) All the 128 pairs of SRAP primers amplified 978 bands,120 pairs (93.8%) of primers were polymorphic and 638 bands (65.2%) were polymorphic. (2) The genetic similarity coefficient of CSTP, CM28TP, MY29TP, NM9TP and their recurrent parents were 0.9346,0.9397,0.9070 and 0.8732, respectively. (3) Cluster analysis revealed that CSTP and CM28TP have least different with the recurrent parent, and was clustered into a small class with the similarity coefficient of 0.93,2. Two forward subtractive cDNA libraries from two pairs of near-isogenic wheat lines, Chuanmai 28 TP (CMTP) and Chinese Spring TP (CSTP), were constructed using suppression subtractive hybridization (SSH). A total of 68 clones in CMTP lines and 197 clones in CSTP lines were identified as potentially overexpressed clones. Thirty-two out of 68 clones in CMTP lines belonged to unknown proteins; while, the remaining 30 clones shared homology to diverse classes of genes involved in protein modulation. and protein synthesis, signal transduction, and ion transporters. Approximately 67% of genes in CSTP lines were either unclassified or had no matches ("no hits") in the database and about 33% of identified genes encoded polypeptides with known functions. Sequence comparisons of cDNA clones between the two forward cDNA libraries revealed that four genes, encoding thioredoxin H, ubiquitin protein ligases, MCM2, and ubiquinol-cytochrome C reductase complex 14 kD proteins, were overexpressed in both libraries. These genes would likely play an important role in determining the three pistils trait in the mutant wheat line.3. The annealing control primer system was used to identify the different expressed genes in three pistils mutation. Using 120 arbitrary ACP primers, we identified 3 differentially expressed genes (DEGs) in young spikes between two near-isogenic lines that are Chuanmai 28 TP (CMTP) and Chinese Spring TP (CSTP) and their recurrent parents. We tentatively designated the three differentially expressed genes as DETP-1, DETP-2 and DETP-3. DETP-1 showed similar function with maize cytoplasmic membrane protein. The cytoplasmic membrane protein involved in cell division in bacteria. DETP-3 is homology to maize endo-1,4-beta-glucanase (EGases). The EGases associated with plant development, cell wall loosening, flowering stem and root expending. DETP-2 showed no significant hit with any sequence found in the database and encodes unknown protein. These genes would likely play an important role in determining the three pistils trait in wheat.4. Full length cDNA sequence of RING domain ubiquitin protein ligase gene TaZFP-1 was acquired by RT-PCR methods. Then, analysis and prediction on the acquired sequences and its amino acids, physicochemical properties, hydrophobicity/hydrophilicity, secondary structure, functional domains, sequence alignment and phylogenetic tree. The results showed that TaZFP-1 gene cDNA was 759 bp in length, encoding 252 amino acids. Molecular weight of the putative TaZFP-1 protein is 27.9 kD with a theoretical PI=6.23. Most amino acids in TaZFP-1 protein are hydrophilic amino acids, so the protein may be a soluble protein. Secondary structure of TaZFP-1 was mainly composed ofα-helices and random coils. Functional domains analysis indicated that the TaZFP-1 is a RING finger domain protein and containing a C3HC4 motif. The molecular evolution threes showed that the TaZFP-1 was clustered into the monocotyledon group and high genetic relationship with O. sativa RING domain E3 ligases.5. The full cDNA sequence of endo-beta-1,4-glucanase gene TaEG was acquired by RT-PCR and RACE methods. Then, analysis and prediction on the acquired sequences and its amino acids, physicochemical properties, hydrophobicity/hydrophilicity, secondary structure, three-dimensional structure, transmembrane domains, sequence alignment and phylogenetic tree. The results showed tha TaEG gene cDNA was 2174 bp in length, encoding 622 amino acids. Molecular weight of the putative TaEG protein is 69.07 kD with a theoretical PI=9.39. Most amino acids in TaEG protein are hydrophilic amino acids, so the protein may be a soluble protein. Secondary structure of TaEG was mainly composed ofα-helices and random coils. Transmembrane domains analysis indicated that TaEG contain a transmembrane domains in 74-96 amino acids, so we prediced the TaEG is a membrane anchored EGase. The cluster analysis also comfirmed that TaEG is a membrane anchored EGase. This study has laid a foundation for further investigate the function of TaEG in wheat pistil development.更多还原