拟南芥CCCH型锌指蛋白OPW（only pollen wall）基因是小孢子发育的关键基因

发布时间：2018-03-26 16:43

  本文选题：拟南芥　切入点：小孢子发育　出处：《上海师范大学》2017年硕士论文

【摘要】：花药发育的第八阶段开始是小孢子从四分体释放出来后单倍体小孢子细胞的发育,单倍体小孢子独立进入花粉囊时,小孢子的花粉壁结构也在着装中,小孢子内出现液泡,之后小孢子细胞内进行两次有丝分裂形成三核花粉粒。这是一个成熟花粉粒的发育过程,如果这一过程里小配子体发育异常,会导致拟南芥的雄性不育。本论文在T-DNA插入突变体Salk_059463株系的群体中,筛选到两株雄性不育突变体,用T-DNA序列上的一对引物对其进行PCR鉴定表明该基因组中没有T-DNA插入。通过背景纯化和遗传分析发现两株雄性不育突变体是由同一单个隐性基因控制的,引起不育的主要原因是在花药发育的第8期开始,小孢子细胞质内容物逐渐减少直至消失,在突变体花药发育的第8期开始对小孢子细胞进行DAPI染色,发现突变体小孢子细胞在第11期没有进行有丝分裂,停留在了单核花粉期;到花药发育的第12期,药室内的小孢子只剩下一个花粉壁空壳,故该突变体命名为opw(only pollen wall),利用图位克隆的方法对OPW基因进行定位,结果表明OPW基因位于第二条染色体上分子标记T28M21和T3G21之间的12Kb区间内,根据TAIR网的数据,该区间内有21个基因注释,通过克隆该区间内的基因并测序,发现opw-1突变体基因组中At2g40140基因编码序列的外显子在第289和第290个碱基之间插入了一个A碱基,而opw-2突变体基因组中At2g40140基因编码序列的外显子在第412和第413个碱基之间插入了一个T碱基,造成编码序列移码使得第424至第426碱基成为终止密码子,故At2g40140是编码OPW的候选基因。进一步根据基因序列设计了互补载体,并转化到农杆菌中,通过农杆菌侵染Ler♂×opw-1♀F1代植株,获得了T0代种子,通过对转基因植株筛选鉴定,得到一定数量的col背景的纯合子可育植株,表示互补成功,初步判定At2g40140为突变基因。根据TAIR网的数据,At2g40140编码的是一个CCCH型锌指蛋白,定位于细胞核内,该蛋白被证明在盐胁迫的途径中起作用,是拟南芥抵御病虫害的CPK介导的钙离子信号通道里CPK3的下游基因。对OPW蛋白进行氨基酸序列分析发现,OPW基因编码的氨基酸序列没有跨膜结构域;进化树分析表明OPW基因与At2g37200有较近的同源关系。
[Abstract]:The eighth stage of anther development was the development of haploid microspore cells after the release of microspore from tetrad. When haploid microspore entered into pollen sac independently, the pollen wall structure of microspore was also in the dressing, and vacuole appeared in microspore. After that, the microspore cells undergo two mitosis to form trinuclear pollen grains. This is a mature pollen grain development process, and if the small gametophyte is abnormal in this process, In this paper, two male sterile mutants were screened in the population of T-DNA inserted mutant Salk_059463, which can lead to male sterility in Arabidopsis thaliana. PCR analysis using a pair of primers on the T-DNA sequence showed that there was no T-DNA insertion in the genome. By background purification and genetic analysis, it was found that the two male sterile mutants were controlled by the same single recessive gene. The main cause of infertility was that at the eighth stage of anther development, the cytoplasmic contents of microspore decreased gradually and then disappeared, and the microspore cells were stained with DAPI at the eighth stage of anther development of mutants. It was found that the mutant microspore cells did not undergo mitosis in phase 11 and remained in the mononuclear pollen stage, and at the 12th stage of anther development, there was only one empty shell of pollen wall left in the microspore chamber. Therefore, the mutant was named opw(only pollen Walla. The mapping method was used to map the OPW gene. The results showed that the OPW gene was located in the 12Kb region between the molecular marker T28M21 and T3G21 on the second chromosome, according to the data of TAIR net. There are 21 gene annotations in this region. By cloning and sequencing the genes in this region, we found that the exon of At2g40140 gene coding sequence in opw-1 mutants inserted an A base between 289 and 290 bases. The exon of the coding sequence of At2g40140 gene in the opw-2 mutants inserted a T base between 412 and 413 bases, resulting in the coding sequence shifting the code sequence to make the 424 to 426 bases as termination codon. Therefore, At2g40140 is a candidate gene encoding OPW. A complementary vector was designed according to the gene sequence and transformed into Agrobacterium tumefaciens to infect Ler through Agrobacterium tumefaciens. 鈾侽pw-1. 鈾，

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