本文選題：擬南芥　切入點(diǎn)：小孢子發(fā)育　出處：《上海師范大學(xué)》2017年碩士論文

【摘要】：花藥發(fā)育的第八階段開始是小孢子從四分體釋放出來(lái)后單倍體小孢子細(xì)胞的發(fā)育,單倍體小孢子獨(dú)立進(jìn)入花粉囊時(shí),小孢子的花粉壁結(jié)構(gòu)也在著裝中,小孢子內(nèi)出現(xiàn)液泡,之后小孢子細(xì)胞內(nèi)進(jìn)行兩次有絲分裂形成三核花粉粒。這是一個(gè)成熟花粉粒的發(fā)育過(guò)程,如果這一過(guò)程里小配子體發(fā)育異常,會(huì)導(dǎo)致擬南芥的雄性不育。本論文在T-DNA插入突變體Salk_059463株系的群體中,篩選到兩株雄性不育突變體,用T-DNA序列上的一對(duì)引物對(duì)其進(jìn)行PCR鑒定表明該基因組中沒有T-DNA插入。通過(guò)背景純化和遺傳分析發(fā)現(xiàn)兩株雄性不育突變體是由同一單個(gè)隱性基因控制的,引起不育的主要原因是在花藥發(fā)育的第8期開始,小孢子細(xì)胞質(zhì)內(nèi)容物逐漸減少直至消失,在突變體花藥發(fā)育的第8期開始對(duì)小孢子細(xì)胞進(jìn)行DAPI染色,發(fā)現(xiàn)突變體小孢子細(xì)胞在第11期沒有進(jìn)行有絲分裂,停留在了單核花粉期;到花藥發(fā)育的第12期,藥室內(nèi)的小孢子只剩下一個(gè)花粉壁空殼,故該突變體命名為opw(only pollen wall),利用圖位克隆的方法對(duì)OPW基因進(jìn)行定位,結(jié)果表明OPW基因位于第二條染色體上分子標(biāo)記T28M21和T3G21之間的12Kb區(qū)間內(nèi),根據(jù)TAIR網(wǎng)的數(shù)據(jù),該區(qū)間內(nèi)有21個(gè)基因注釋,通過(guò)克隆該區(qū)間內(nèi)的基因并測(cè)序,發(fā)現(xiàn)opw-1突變體基因組中At2g40140基因編碼序列的外顯子在第289和第290個(gè)堿基之間插入了一個(gè)A堿基,而opw-2突變體基因組中At2g40140基因編碼序列的外顯子在第412和第413個(gè)堿基之間插入了一個(gè)T堿基,造成編碼序列移碼使得第424至第426堿基成為終止密碼子,故At2g40140是編碼OPW的候選基因。進(jìn)一步根據(jù)基因序列設(shè)計(jì)了互補(bǔ)載體,并轉(zhuǎn)化到農(nóng)桿菌中,通過(guò)農(nóng)桿菌侵染Ler♂×opw-1♀F1代植株,獲得了T0代種子,通過(guò)對(duì)轉(zhuǎn)基因植株篩選鑒定,得到一定數(shù)量的col背景的純合子可育植株,表示互補(bǔ)成功,初步判定At2g40140為突變基因。根據(jù)TAIR網(wǎng)的數(shù)據(jù),At2g40140編碼的是一個(gè)CCCH型鋅指蛋白,定位于細(xì)胞核內(nèi),該蛋白被證明在鹽脅迫的途徑中起作用,是擬南芥抵御病蟲害的CPK介導(dǎo)的鈣離子信號(hào)通道里CPK3的下游基因。對(duì)OPW蛋白進(jìn)行氨基酸序列分析發(fā)現(xiàn),OPW基因編碼的氨基酸序列沒有跨膜結(jié)構(gòu)域;進(jìn)化樹分析表明OPW基因與At2g37200有較近的同源關(guān)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，

本文編號(hào)：1668690