【作者】 陈起振；

【导师】 曹家树；

【作者基本信息】 浙江大学 ， 蔬菜学， 2011， 硕士

【摘要】 芸薹属是十字花科植物的重要类群,也是我国栽培面积最大的蔬菜作物类群。白菜(Brassica campestris L. ssp. chinensis Makino)是芸薹属芸薹种的一个亚种,是典型的异花授粉作物,具有显著的杂种优势,F0代杂种的推广应用可以提高白菜的生产水平。雄性不育材料在白菜F1代种子的生产中具有重要地位,其选育和利用日益受到人们的重视。以白菜雄性不育材料为基础,分析白菜花发育相关基因的表达特征,分离花发育相关基因并验证基因的功能,不仅可以促进白菜雄性不育材料的选育,而且对于揭示植物花发育的分子机制也具有重要的理论意义。本实验室采用ATHl芯片分析了授粉受精前后雌蕊基因的表达差异,发现At2g39060在授粉后的雌蕊中上调表达,同时我们也发现该基因在白菜mmc对应的野生型中上调表达。At2g39060编码的蛋白质是一个MtN3/saliva家族蛋白,该基因在拟南芥中的功能还是未知。为了认识该基因的结构和表达特征,为进一步研究其功能打下基础,本研究利用cDNA末端快速扩增(rapid amplification of cDNA ends, RACE)技术和同源扩增的方法克隆基因全长,分析其序列特征并预测其蛋白质结构和功能,进而采用实时定量聚合酶链式反应(real-time quantitative polymerase chain reaction, qRT-PCR)分析其在可育株和不育株的不同发育阶段的花蕾中和营养器官中的表达情况,采用组织原位杂交进行该基因的细胞定位,预测其与花发育的关系,同时构建反义RNA表达载体,通过农杆菌介导法转化菜心植株,比较分析转基因植株和对照植株在形态、育性、花器结构、花粉形态及萌发能力等方面的差异,为阐明该基因的生物学功能提供实验证据。取得的主要成果如下：(1)从ATH1基因芯片获得的一个在白菜核雄性不育两用系’Bcajh97-01A/B’可育株和授粉后雌蕊中同时上调表达的基因At2g39060入手,利用同源扩增和RACE技术成功克隆出它在白菜中的同源基因cDNA全长,命名为白菜蜜腺雄蕊发育基因(Brassica campestris Nectary Stamen, BcNS)。该基因的DNA全长为1603碱基对(base pair, bp), cDNA全长为1069bp,利用ClustalX软件比较该基因的cDNA和DNA序列,发现该基因含有6个外显子和5个内含子,内含子长度分别为188 bp、84bp、92 bp、94 bp和76 bp,6个外显子的长度分别为49 bp、37 bp、211bp、162 bp、120 bp和234 bp,外显子-内含子交接处符合GT-AG规律,符合典型的植物内含子剪切模式。BcNS的cDNA序列中,最大开放阅读框(Open reading frame, ORF)为813bp,起始密码子ATG始于第16位,终止密码子TGA止于第828位,5’和3’非翻译区分别为15 bp和241 bp,预测该基因可编码一个含有270个氨基酸的蛋白质。BcNS蛋白质分子量为30110.08 Daltons,等电点为9.117,pH 7.0时的电荷为7.453,包含碱性氨基酸(K、R)21个,酸性氨基酸(D、E)14个,疏水氨基酸(A、I、L、F、W、V)124个,极性氨基酸(N、C、Q、S、T、Y)70个。该蛋白具有2个N-糖基化位点,1个依赖于cAMP和cGMP的蛋白激酶磷酸化位点,1个酪蛋白激酶Ⅱ磷酸化位点,2个N-豆蔻酰化位点,4个蛋白激酶C(protein kinase C, PKC)磷酸化位点,1个Big-1(bacterial Ig-like domain 1)结构域位点,2个MtN3/saliva家族位点。该基因的推测蛋白在N端由一段信号肽,信号肽和成熟肽之间的可能切割位点在第26和第27个氨基酸之间,有7个跨膜区,PSORT程序定位该基因在细胞内该蛋白为分泌途径蛋白的可能性最高。(2)实时定量聚合酶链式反应(qRT-PCR)和原位杂交技术分析BcNS的时空表达表明,该基因用在’Bajh97-01A/B’不育株和可育株的五级花蕾和开放的花中检测到了该基因的表达,开放花中的表达量明显高于第5级花蕾,其它时期没有检测到表达。另外,该基因主要在不育株与可育株的蜜腺和雄蕊中表达,在花瓣和萼片中的表达量极低,在雌蕊中没有表达。其中,该基因在不育株与可育株的雄蕊中的表达量相差无几,几乎相等,但是表达量都不高。而在蜜腺中的表达量非常高,大约是雄蕊中表达量的26～47倍,同时,该基因可育株中蜜腺的表达量大约是不育株中的1.7倍。花器官中的表达情况说明该基因在蜜腺和雄蕊中特异表达,但是不育系和可育系中的表达量差别也并不大,说明该基因与白菜的育性并没有直接的关系,而可能在其他方面起着一定的作用。但是原位杂交只在蜜腺检测到了该基因的表达,原因可能是原位杂交这种技术的局限性。(3)构建了反义RNA表达载体pBI35S-BcNS,农杆菌介导法转化菜心,获得了Kan抗性菜心植株。分子检测表明己得到了转基因植株,对转基因植株的生长状况、花器官发育等进行观察,发现抑制BcNS的表达会影响植株花器官的发育,因此推论BcNS是一个与白菜花发育相关的新基因。更多还原

【Abstract】 Brassica is one of the most important genus of Cruciferae, and is one of the vegetable crops cultured most widely in Chinese cabbage pak-choi (Brassica campestris L. ssp. chinensis Makino), a subspecies of Brassica campestris L, is a typical allogamy plant with significant heteosis, and the popularization and application of its Fl hybrid seeds can improve the level of production in Chinese cabbage. Earlier study in our laboratory has found that At2g39060 was up-regulated in the fertile B line of the Chinese cabbage-pak-choi (Brassica campestris L. ssp. chinensis Makino) genic male sterile AB line (Bcajh97-01A/E) based on an ATH1 gene chip. The protein At2g39060 encoded belongs to MtN3/saliva family, and the function of At2g39060 is unknown. In order to realize the structure and expression partern of this gene, we cloned its full length cDNA using homologous gene amplification and rapid amplification of cDNA ends (RACE). And then, we analyzed its sequence characteristics and predicted protein structure. Real time quantitive polymerase chain reaction (qRT-PCR) and in situ hybrization were performed to inverstigate the expression parttern of the gene during different stages of plant development and forecast the gene’s function. Then, the antisense RNA expression vector of BcNS were constructed, and transfomed into flowering Chinese cabbage (B. campestris ssp. chinensis var. parachinensis Tsen et Lee) by agrobacterium-mediated method to obtain their loss-of-function mutants for confirming its function. The major study results were as follows:(1)We cloned the homologous gene of At2g39060 in Chinese cabbage-pak-choi based on a ATH1 gene chip, using homologous gene amplification and RACE.Then we named it Brassica campestris Nectary Stamen (BcNS). The lengths of cDNA and DNA of BcNS are 1603bp and 1069bp. Its DNA includes six extrons and five introns, and the lengths of extons are 49 bp,37 bp,211 bp,162 bp,120 bp,234 bp; the lengths of introns are 188 bp,84 bp,92 bp,94 bp,76 bp, respectively. The open reading frame of its cDNA is of 813bp, encoding 270 amino acid residues. The molecular weight of BcNS is 30110.08 Daltons, and its isoelectric point is 9.117. BcNS includes two N-glycosylation sites, one cAMP- and cGMP-dependent protein kinase phosphorylation site, one Casein kinaseⅡphosphorylation site, two N-myristoylation sites, four Protein kinase C phosphorylation sites, one Big-1 (bacterial Ig-like domain 1) domain profile, two MtN3/saliva family sites.(2) Real time quantitive polymerase chain reaction (qRT-PCR) and in situ hybrization were performed to inverstigate the expression parttern of BcNS. we detected the expression of this gene in the sterile plants and fertile plants of ’Bajh97-01A/B’ of the level five flower buds and opening flowers, and the expression in open flowers was significantly higher than level five buds, but at other stages of buds no expression was detected. In addition, the gene mainly express in the nectaries and stamens of the sterile plants and fertile plants, while the expression in the petals and sepals are very low, and no expression exsits in the pistil. Expressions of BcNS in stamens of the sterile plants and fertile plants are almost the same, but the expressions are not high. BcNS is highly expressed in the nectary, and the expression quantity is about 26～47 times higher than that in stamens.And the gene expression in the nectary of the fertile plants is about 1.7 times as that in sterile plants. The expression of BcNS in floral organs shows that the gene specifically expressed in the nectaries and stamens, but expressions between the sterile and fertile lines are not significant different. This indicates that the gene has no direct relationship with the fertility of Chinese cabbage, and it may play a role in other areas. However, expression of the gene detected only in the nectary by in situ hybridization, and this may be due to the limitations of this technology.(3) Anti-sense plant expression vector pBI35S-BcNS was constructed, and transformed into flowering Chinese cabbage by agrobacterium-mediated method. The molecular assay showed that pBI35S-BcNS constructs were integrated into the genomes of BcNS transgenic plants with Kanamycm resistance. Growth performance, floral organ development of transgenic plants were observed, these results showed that floral development was influenced by down-regulated expression of BcNS in transgenic plants. These results suggest that, BcNS is a new flower development-related gene in Chinese cabbage.更多还原