Study of anther specific genes of petunia and rice by transposon mediated gene disruption or transformation mediated gene silencing

通过转座子介导的基因破坏或转化介导的基因沉默研究矮牵牛和水稻的花药特异性基因

基本信息

批准号：
14560001
负责人：
KUBOYAMA Tsutomu
金额：
$ 2.37万
依托单位：
Ibaraki University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14560001/
关键词：
pollen anther sterile petunia transposon mediated gene disruption β-1,3 glucanase cDNA microarray rice 花粉管雄生不稔

项目摘要

Two β-1,3 glucanase genes (PhPOGLU, PhPTGLU) found in EST of petunia pollen were targeted for transposon mediated gene disruption. 1024 plants of W138 line were screened by 3 dimensional PCR (Koes et al. 1995). One dTph1 insertion mutant of PhPOGLU was found. This mutant should lose the glucanase activity because dTph1 was inserted in 56th codon of PhPOGLU and this position was relatively near the aminoterminal of PhPOGLU (491 a.a.). However, this mutant did not show any phenotypic change in pollen tube elongation. Pollen fertility was decreased in the, mutant (68.4%), but heterozygous plants showed severer pollen sterility (ca. 30%) than homozygous mutants. Therefore, it is not clear whether the cause of this decreased pollen fertility is due to the mutation of PhPOGLU or not. When we made plant population, some pink flowered female sterile plants (pfs line) were observed. The plants of pfs line had an1 as hetero zygous. One was an1 which had dTphl insertion in the intron exon junction (an1-W138) and the other was an1 which had a 5bp footprint remained after excision of dTphl from an1-W138 allele (an1-pfs). This hererozygous state should be the cause of the female sterile because plants that had homozygous an1-pfs or homozygous an1-w138 did not show female sterility.In addition, we performed the analyses of the cDNA microarray to monitor gene expression patterns during anther development in rice. Microarray analysis of 4,304 cDNA clones revealed that 259 cDNA clones (156 non redundant groups) were specifically or predominantly expressed in anther tissues and were regulated by developmental stage-specific manners in the anther tissues. These co-regulated genes would be important for development of functional anther tissues. Furthermore, we selected several clones for RNA in situ hybridization analysis and transformation mediated gene silencing. Four lines derived from two chimeric gene constructs show male sterility.

在矮牵牛花粉中发现的两个β-1,3葡萄糖酶基因（Phpoglu，phptglu）的靶向是转座子介导的基因破坏。通过3维PCR筛选了1024种W138系的植物（Koes等，1995）。发现一个DTPH1插入突变体的phpoglu。该突变体应失去葡萄糖酶活性，因为将DTPH1插入了第56个Phpoglu的密码子中，并且该位置相对靠近phpoglu的aminoterminal（491 A.A.）。但是，该突变体没有显示花粉管伸长的任何表型变化。突变体（68.4％）的花粉生育能力降低，但杂合植物比纯合突变体显示出多种花粉不育（约30％）。因此，尚不清楚这种花粉生育能力降低的原因是否是由于phpoglu的突变。当我们制造植物种群时，观察到一些粉红色的雌性无菌植物（PFS系）。 PFS系的植物具有AN1为异性。一个是AN1，它在内含子外显子连接（AN1-W138）中插入DTPHL，另一个是AN1，其在从AN1-W138等位基因（AN1-PFS）切除后，其占5BP足迹保留。这种遗传性状态应该是雌性无菌的原因，因为具有纯合AN1-PFS或纯合子AN1-W138的植物没有表现出女性的不育。此外，我们对大米的其他发育过程中的基因表达模式进行了分析以监测基因表达模式。对4,304个cDNA克隆的微阵列分析表明，259个cDNA克隆（156个非冗余组）是在其他组织中特异性或主要表达的，并受到其他组织中发育阶段特异性的方式调节。这些共同调节的基因对于开发功能性A组织很重要。此外，我们选择了几个克隆来进行RNA原位杂交分析和转化介导的基因沉默。源自两个嵌合基因构建体的四条线显示男性不育。