本文關(guān)鍵詞：水稻OsCYP2及其互作蛋白OsZFP調(diào)控側(cè)根生長的功能研究　出處：《浙江大學(xué)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： OsCZP2 水稻 側(cè)根生長 生長素 蛋白互作 OsZFP

【摘要】：側(cè)根是水稻胚后發(fā)育的重要器官,在水稻根的構(gòu)型建立中起著舉足輕重的作用。盡管前人的研究報(bào)道中已對水稻側(cè)根的形態(tài)和生理作用有了較詳盡的描述,但是對于控制水稻側(cè)根形成的相關(guān)基因所知仍然不多,因此對水稻側(cè)根發(fā)生機(jī)制的研究及關(guān)鍵基因的克隆具有重要意義。本研究以水稻品種'愛知旭'野生型(WT)、OsCYP2基因過表達(dá)和干擾三種基因型植株為材料,通過對其表型、表達(dá)譜驗(yàn)證以及互作蛋白OsZFP的基因克隆和功能分析,研究了 OsCZP2和OsZFP兩個基因在生長素信號通路上對側(cè)根發(fā)生的影響,取得創(chuàng)新性結(jié)果如下:1、OsCYP2是一個在水稻各組織器官中廣泛表達(dá)的基因,其干擾之后導(dǎo)致側(cè)根形成受阻,側(cè)根數(shù)量顯著減少。GUS染色結(jié)果顯示,穎殼、葉、莖和根等組織部位均呈現(xiàn)明顯的藍(lán)色信號,其中穎殼顏色最深,表明OsCYP2的啟動子為組成型表達(dá)啟動子。外源生長素IAA處理前后,OsIAA1、OsIAA8和OsIAA31基因的表達(dá)量在OsCYP2干擾植株中無顯著性差異,表明其依賴于OsCYP2信號途徑。并且,除OsIAA1和OsIAA20,OsCZP2-RNAi植株中其余4個生長素響應(yīng)基因(OsIAA8、11、23和31)的誘導(dǎo)表達(dá)量與WT相比均顯著下降,表明OsCZP2干擾植株對生長素處理存在一定的不敏感性。2、RNA-seq結(jié)果顯示,與WT相比,OsCYP2-RNAi的31515個差異表達(dá)基因中有3158個表現(xiàn)顯著性差異,其中1267個基因呈上調(diào)趨勢,1891個呈下調(diào)趨勢。11個顯著差異表達(dá)基因與側(cè)根形成有關(guān),4個差異基因與生長素有關(guān)。RT-PCR驗(yàn)證結(jié)果表明以上相關(guān)基因的上/下調(diào)趨勢與RNA-seq結(jié)果一致,由KEGG數(shù)據(jù)庫分析得到差異表達(dá)基因的前20個富集pathway中核糖體富集最顯著,其次是次生代謝生物合成和類黃酮合成。3、構(gòu)建了水稻cDNA文庫,通過酵母雙雜交篩選,獲得3個陽性互作蛋白,之后經(jīng) GST-pulldown 體外互作驗(yàn)證,OsCYP2 與 OsZFP(Os01g0252900)存在互作。經(jīng)同源性比對,OsZFP屬CCHC型鋅指結(jié)構(gòu)蛋白;聚類分析表明,在擬南芥、大麥、高粱和玉米中高度保守,而在人類、酵母和小麥沒有此結(jié)構(gòu)域。4、利用反向遺傳學(xué)方法,對水稻OsZFP基因調(diào)控側(cè)根生長發(fā)育的生物學(xué)功能進(jìn)行研究。以水稻品種'愛知旭'野生型為試驗(yàn)材料,構(gòu)建了 PCAMBIA1300-OsZFP-RNAi表達(dá)載體并獲得多個穩(wěn)定遺傳轉(zhuǎn)化株系。結(jié)果表明,T2代OsZFP-RNAi植株同樣出現(xiàn)水稻側(cè)根形成受阻,側(cè)根缺失的表型。之后構(gòu)建OsZFP基因與'GFP融合的表達(dá)載體并進(jìn)行水稻原生質(zhì)體轉(zhuǎn)化,亞細(xì)胞定位結(jié)果表明OsZFP蛋白定位于細(xì)胞核。外源生長素IAA處理下,RT-PCR結(jié)果顯示OsZFP-RNAi植株中六個Aux/IAA基因(OsIAA1、2、6、20、23和24)與無激素處理相比沒有顯著差異,表明OszFP基因被干擾之后,植株的生長素信號途徑受到影響。
[Abstract]:The lateral root is an important organ development after rice embryo, in rice root configuration plays an important role in the establishment. Although previous research reports have been on rice lateral root morphological and physiological function of a more detailed description, but the genes controlling rice lateral root formation is still not known, so cloning research and the key gene on pathogenesis of rice root has important significance. In this study, rice cultivar 'Aichi Asahi' wild type (WT), OsCYP2 gene expression and interference of three genotypes as materials, based on their phenotype, expression and verification of interaction protein OsZFP gene cloning and functional analysis of OsCZP2 and OsZFP two genes in auxin signaling pathway on lateral root formation, the innovative results are as follows: 1, OsCYP2 is a widely expressed in various tissues and organs of rice genes, after its interference Lead to lateral root formation is blocked, the number of lateral roots was significantly reduced..GUS staining showed that glume, leaf, stem and root tissues showed a clear signal to the blue part, wherein the glume color deep, showed that the OsCYP2 promoter for constitutive expression promoter. The exogenous auxin IAA before and after the treatment, OsIAA1, expression of OsIAA8 and the OsIAA31 gene had no significant difference in plant OsCYP2 interference, showed that the OsCYP2 dependent signaling pathway. And, in addition to OsIAA1 and OsIAA20, the remaining 4 auxin response genes in OsCZP2-RNAi mutants (OsIAA8,11,23 and 31) induced expression of WT decreased significantly, OsCZP2 interference showed that plants on auxin.2 sensitivity the RNA-seq results showed that compared with WT, the 31515 difference expression of OsCYP2-RNAi 3158 showed significant differences in gene, of which 1267 genes were up-regulated and 1891 were downregulated.11 The formation of a gene with the roots significantly differentially expressed genes and auxin, 4 different.RT-PCR validation results showed that the above genes / downward trend was consistent with RNA-seq, KEGG database by analysis of gene expression difference obtained before the 20 enrichment of pathway ribosomes in the enrichment was most significant, followed by secondary metabolism and biosynthesis of flavonoid biosynthesis.3 construction of rice cDNA library by yeast two hybrid screening, 3 positive interaction protein after GST-pulldown in vitro interaction test, OsCYP2 and OsZFP (Os01g0252900) interaction. By homology, OsZFP belongs to CCHC type zinc finger protein; cluster analysis showed that in Arabidopsis, barley, sorghum and maize highly conserved in human, yeast and wheat without the.4 domain, using the method of reverse genetics, biological function of rice OsZFP gene regulation of lateral root growth and development To study. With the rice variety 'Aichi Asahi' wild type as the experimental materials, the expression vector PCAMBIA1300-OsZFP-RNAi was constructed and a stable genetic transformation strain. The results showed that the T2 generation of OsZFP-RNAi plants also appear rice lateral root formation is blocked, root missing. After constructing the phenotypic expression vector of OsZFP gene and'GFP fusion and rice protoplast transformation, subcellular localization results showed that OsZFP protein was localized in the nucleus. The exogenous auxin IAA, RT-PCR results showed that six Aux/IAA genes in OsZFP-RNAi mutants (OsIAA1,2,6,20,23 and 24) and non hormone treatment had no significant difference compared with that after OszFP gene was interfered, the auxin signaling pathway was affected.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：S511

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本文編號：1411869