【作者】 栗现芳；

【导师】 张改生；

【作者基本信息】 西北农林科技大学 ， 遗传学， 2011， 博士

【摘要】 多子房小麦具有明显的穗粒数优势,如能应用到杂交小麦育种中将会是提高繁制种系数的一种有效途径,进而对杂交小麦的发展将会起到一个极大地促进作用。目前有关多子房小麦的研究多集中在花器官的发育过程、种子萌发过程中的生化研究、分子标记、基因定位和遗传分析,对小麦多子房性状形成的分子机制研究至今仍是空白。小麦多子房品系Ⅱ(简称多Ⅱ)近等基因系(near-isogenic line, NIL)是由西北农林科技大学专为研究多子房性状的分子遗传机理而创制的一种试验材料,其多子房、单子房性状遗传稳定,其它遗传背景和农艺性状与轮回亲本一致,是研究多子房性状分子机制的良好试材。为了深入揭示小麦多子房性状形成的分子机制,本研究利用上述材料,首次以SSH(Suppression Subtractive Hybridization, SSH)技术分别构建了多子房和单子房相关基因特异表达的抑制消减杂交cDNA文库,通过对两个cDNA文库的克隆测序、EST序列比对分析和功能分类,探讨了小麦多子房和单子房性状各自表达基因的种类和功能的异同,并从中选择了部分基因进行了表达分析和cDNA、DNA序列的克隆,获得如下重要结果:1.对小麦多ⅡNIL的外显率、多粒结实率、发芽率进行了调查与分析,同时在田间调查过程中发现了每朵小花具有4～6个雌蕊的新变异类型。不同株系间多子房性状外显率分布在77.2～95.3%之间,平均为88.6%;多粒种子平均结实率为79.9%;多子房株系主位种子与单子房株系种子发芽率基本一致,副位种子发芽率稍低;分析推测小麦多子房新突变现象是在原本遗传稳定的多子房性状基础上进一步发生了雄蕊心皮化的结果,第4～6个雌蕊未能成功受精结实。2.以小麦多ⅡNIL的多子房和单子房株系幼穗cDNA互为Tester和Driver,利用SSH技术成功构建了小麦多子房和单子房性状相关基因的cDNA文库(简称为Mu文库和Mo文库)。Mu文库共得到约2536个菌落,其中白斑2383个,重组率为93.97%;Mo文库共得到约4543个菌落,其中白斑4359个,重组率为95.95%。分别采用巢式引物和通用引物对随机挑取的克隆进行PCR检测,两个文库85%以上的克隆皆能扩增出目的片段,基本介于200～750bp之间,多数集中在400～500 bp之间。对部分阳性克隆提取质粒及酶切,插入片段大小主要集中在450bp左右,与菌液PCR检测结果一致。3.从Mu和Mo文库中,各随机选择100个阳性克隆进行测序。Mu文库中所获EST片段平均大小为378bp。去除低质量序列及重复序列后,共获得90条EST,其中65条功能已知,占全部EST的72.22%。参与蛋白合成的最多,占27.69%;其次为亚细胞定位相关的基因,占10.77%;与能量、转录相关的基因各为7.69%;与新陈代谢、生长发育、结合功能蛋白相关的基因各为6.15%;占比例较低的是与蛋白加工、细胞通信与信号转导、细胞防御等相关的基因。Mo文库中所获EST片段平均大小为339bp。去除重复序列后,共获得89条EST,其中60条功能已知,占全部EST的67.42%。在已知功能序列中,未分类功能蛋白的序列较多,占33.33%;与新陈代谢有关的基因占11.67%;亚细胞定位相关基因占10%;与能量、蛋白合成、蛋白加工、结合功能蛋白相关的基因各占6.67%。不同功能的蛋白在两个库中所占比例不一致,Mu文库以蛋白合成、转录和生长发育相关蛋白为主;而Mo文库以物质代谢、蛋白加工及细胞运输途径相关蛋白为主。4.利用半定量RT-PCR对两个文库中的12个相关基因进行了时空表达研究。Mu文库中所选的6个基因在多子房株系都有不同程度的上调表达,40S核糖体蛋白S6、核内小核糖核蛋白质G、核糖体蛋白L10A差异较为明显,其次为核糖体蛋白s15a、14-3-3蛋白、60s核糖体蛋白L21,而假定的脯氨酰4 -羟化酶、伴侣蛋白、CER1蜡质合成酶基因差异不明显;Mo文库中尿苷二磷酸葡糖醛酸脱羧酶、60S核糖体蛋白L17-1在单子房株系中上调表达,半胱氨酸蛋白酶抑制剂差异较小;多数基因1~12mm幼穗时期内表达量呈上升趋势,广泛存在于各种组织,在茎顶端表达量高于其他组织。研究推测,差异表达的基因可能参与了多子房性状的发生。5.以SSH文库中筛选出的小麦RPS15a和RPL21基因序列为信息探针,通过同源序列搜索、聚类拼接、RT-PCR和PCR克隆,获得了RPS15a、RPL21基因的cDNA和DNA序列。小麦RPS15a基因的cDNA序列(GenBank登录号:HM055513)长为413 bp,编码130个氨基酸;DNA序列(GenBank登录号:HM063421)长为642 bp,含有3个外显子和2个内含子。小麦RPL21基因cDNA序列(GenBank登录号:HM138480)长521bp,编码164个氨基酸,氨基酸序列在植物与动物间相似性较差;DNA序列(GenBank登录号:HM138481)长1600bp,含有2个外显子和1个内含子。在不同材料中,这两个核糖体蛋白基因在多子房株系2～4mm幼穗中表现不同程度的表达上调,可能与小麦多子房性状的发生有关,其具体功能作用尚需进一步验证。6.以SSH文库中筛选出的小麦Cab基因序列为信息探针,通过同源序列搜索、聚类拼接和RT-PCR,获得了小麦Cab基因的cDNA序列(GenBank登录号:HM362991)。序列长862bp,编码266个氨基酸,与之前EST序列拼接延伸后得到924bp的序列。在不同材料中,Cab基因在单子房株系2～3和3～4mm中表达量下调;在单子房株系1～6mm幼穗时期内,2～3mm幼穗中表达量较低;不同组织中差异明显,幼叶中表达量最高,幼根中无表达。更多还原

【Abstract】 Due to the unique characteristics of the floral organ and with multiple ovaries, the multi-ovary wheat can enhance the propagation coefficient and play a important role in the development of hybrid wheat. Previous research has focused on the development of floral organs, biochemical analysis of seed germination and molecular markers, as well as gene localization and genetic analysis. About the molecular mechanism of the formation of multi-ovary trait is needed to study at present. The NIL of multi-ovay wheat DuoⅡshowed the same genetic background except for the ovary character. Therefore, it was an excellent material for studying the formation mechanism of multi-ovary trait in wheat. In this study, the materials mentioned earlier were initially employed to construct two cDNA libraries of multi-ovary and mono-ovary trait by suppression subtractive hybridization (SSH). Through sequencing, alignment analyses and functional annotation of the expressed sequence tags (ESTs), the types of expressed genes and gene functions have been compared with the 2 libraries. The expression analysis (different materials, different stages and different organs) and clone of partial gene was researched. We hope to give some explanations to the molecular mechanism of the formation of multi-ovary trait. The results were listed as follows:1. The penetrance, maturing rate and percentage germination of the NIL of the multi-ovary wheat DuoⅡwas investigated and analyzed. At the same time, the phenomenon of 4~6 pistil in one floret was found during the process of investigation in the field. The penetrance was 77.2～95.3% in different multi-ovary lines and the average was 88.6%. The average of maturing rate of multiple seeds was 79.9%. The percentage germination of the seeds on main location of multi-ovary line and the seeds of mono-ovary line was no difference. The percentage germination of the seeds on additional location of multi-ovary line was lower than the seeds of mono-ovary line. The new mutation phenomenon of multi-ovary wheat was a result of carpelloid stamens (the stamens changed into multi-pistils) though analysis. The 4~6 pistil cannot be fertilized.2. The multi-ovary line and mono-ovary line of the NIL were employed to construct two cDNA libraries related to multi-ovary (Mu) and mono-ovary (Mo) by SSH. About 2536 clones were obtained from Mu library and there were 2383 positive clones, the recombination frequency was 93.97%. About 4543 clones were obtained from Mo library and there were 4359 positive clones, the recombination frequency was 95.95%. Random differentially expressed cDNA clones were screened by PCR (Nest primer or M13 primer) amplification in the two subtraction libraries. More than 85% of the clones contained an insert and while most of the cDNA inserts were 400~500 bp, the range of clones extended from more than 250 bp to less than 750 bp. Though extraction of plasmid and Enzyme digested of some positive clones, the inserted fragment mainly concentrated approximately 450bp. This results was similar to PCR.3. One hundred positive clones were randomly selected and sequenced from the Mu library and Mo library respectively. The average size of EST in Mu library was 378bp. Ninety EST sequences were obtained after removal of low-quality and repeated sequences. Sixty-five ESTs (72.22% of the 90 ESTs) had translated sequences homologous to proteins with a known function. Among the sixty-five ESTs, 27.69% of them were categorized into protein synthesis, whereas, 10.77% of them were involved in subcellular localization, 7.69% of them were related to energy and transcription respectively, 6.15% of them were related to metabolism, development and protein with binding function respectively, and a small proportion of ESTs was related to protein fate, signal transduction mechanism and cell defense. The average size of EST in Mo library was 339bp. Eighty-nine EST sequences were obtained after the removal of the repeated sequences. Sixty ESTs, accounting for 67.42%, had sequence similarity with known function proteins. Among the sixty EST sequences, 33.33% were unclassified protein. 11.67% of the annotated ESTs were associated with metabolism, 10% of them were related to the subcellular localization, and 6.67% of them were in relation to the energy, protein synthesis, protein fate and protein with binding function respectively. The proportion of different functional proteins was inconsistent between the two libraries. Proteins related to protein synthesis, transcription and development were found frequently in the Mu library. Some proteins associated with metabolism, protein fate and cellular transport were found frequently in the Mo library.4. The expression patterns of 12 genes from two library were analyzed by RT-PCR. The results showed that six genes from Mu library were up-regulated by a different extent in multi-ovary wheat. The expression differences of 40S ribosomal protein S6, snRNP G and ribosomal protein L10A were more significant than those of ribosomal protein S15A , 14-3-3 protein and 60s ribosomal protein L21. Different expression of putative CER1, chaperonin and putative prolyl 4-hydroxylase were not significantly different. In contrast, expression analysis of genes from the Mo library showed that UDP-D-glucuronate decarboxylase and ribosomal protein L17-1 were up-regulated, while the expression level of cysteine proteinase inhibitor showed no significant difference. Most genes’expression presented an up-regulated tendency at the 1~12mm development stages of young ear. The examined genes were abundantly expressed in different tissues and the quantity of expression for most of them was highest in stem shoot meristem. Study speculated that differential expression genes may be involved in the occurrence of multi-ovary trait.5. The EST highly similar to RPS15a and RPL21 gene from the SSH library was used as a query probe to blast the Genbank databases. Based on the assembled homologous cDNA sequence, both cDNA and DNA sequences encoding RPS15a and RPL21 were isolated and characterized by RT-PCR and PCR. The cDNA sequence of RPS15a in wheat (GenBank accession No.HM055513) was 413 bp in length and the open reading frame encoded a peptide of 130 amino acids. The DNA sequence of RPS15a (GenBank accession No.HM063421) was 642 bp in length, which contained three extrons and two introns. The cDNA sequence of RPL21 (GenBank accession No.HM138480) was 521bp in length and the open reading frame encoded a peptide of 164 amino acids. The homology of amino acids of RPL21 was not conserved between the plants and animals. The DNA sequence of RPL21 (GenBank accession No.HM138481) was 1600bp in length, which contained two extrons and one intron. According to expression analysis, the expression quantity of the RPS15a and RPL21 gene in 2～4 mm ears of multi-ovary line was higher than that in mono-ovary line. They are may be related to the occurrence of multi-ovary trait. Their function was needed to more study.6. The EST highly similar to Cab gene from the SSH library was used as a query probe to blast the Genbank databases. Based on the assembled homologous cDNA sequence, the cDNA sequence encoding Cab were isolated and characterized by RT-PCR. The cDNA sequence of Cab (GenBank accession No.HM138480) was 862bp in length and the open reading frame encoded a peptide of 266 amino acids. The cDNA sequence of Cab and the EST highly similar to Cab gene from the SSH library was assembled and then the cDNA sequence of Cab became 924bp in length. The expression quantity of Cab gene in 2~3 and 3~4mm ears of mono-ovary line was lower than that in multi-ovary line. The expression of Cab gene in 2~3mm ears was lower than that in other stages in 1~6mm ears. The expression quantity of Cab gene was highest in young leaves and no detectable in young roots.更多还原