玉米中ZmPIFs基因的克隆與功能分析
發(fā)布時(shí)間：2018-08-13 16:22
【摘要】：光對(duì)植物的生長(zhǎng)發(fā)育起著重要的作用,一方面它不僅為植物帶來(lái)了能量,使植物通過(guò)光合作用完成了同化物的積累;另一方面光又扮演了信號(hào)分子的角色,使植物通過(guò)感應(yīng)光的變化調(diào)整自身的生長(zhǎng)發(fā)育。植物通過(guò)體內(nèi)的光敏色素(phytochrome,PHY)感知光信號(hào),并通過(guò)光敏色素互作因子(phytochrome-interacting factors,PIFs)與光敏色素的相互作用調(diào)節(jié)植物的生長(zhǎng)過(guò)程。PIFs對(duì)擬南芥的生長(zhǎng)發(fā)育起著至關(guān)重要的作用,促進(jìn)下胚軸的伸長(zhǎng),并抑制子葉的張開(kāi),還有抑制光形態(tài)建成。目前對(duì)于PIFs的研究主要集中于模式植物,而對(duì)一些高等作物尤其是玉米中ZmPIFs的研究還較少。因此,對(duì)玉米中ZmPIFs基因家族的研究有著重要的意義。在本實(shí)驗(yàn)中,我們從玉米基因數(shù)據(jù)庫(kù)MaizeGDB中確定了玉米ZmPIFs基因家族的成員,探究了該基因家族的表達(dá)模式,分離得到了10個(gè)玉米基因。將其中4個(gè)基因轉(zhuǎn)入擬南芥,結(jié)果發(fā)現(xiàn)bHLH44基因在抑制光形態(tài)建成和影響植株響應(yīng)重力方面具有重要作用。主要結(jié)果如下:(1)根據(jù)NCBI上查詢(xún)的擬南芥AtPIFs基因家族序列,將其在MaizeGDB上進(jìn)行BLAST分析,獲得了17個(gè)玉米ZmPIFs同源基因。通過(guò)進(jìn)化樹(shù)比對(duì)、基因序列分析、氨基酸序列分析和蛋白結(jié)構(gòu)域預(yù)測(cè),初步分析、預(yù)測(cè)了玉米ZmPIFs基因的結(jié)構(gòu)與功能。最終確定了10個(gè)與擬南芥AtPIF1,AtPIF3,AtPIF4和AtPIF5同源且起主要作用的玉米ZmPIFs基因:ZmbHLH12、ZmbHLH30、ZmbHLH37、ZmbHLH40、ZmbHLH44、ZmbHLH51、ZmbHLH74、ZmbHLH88、ZmbHLH113和ZmbHLH180。由于PIFs基因?qū)儆赽HLH基因家族,所以我們對(duì)玉米ZmPIFs基因的命名使用了其在bHLH基因家族中的編號(hào)。實(shí)時(shí)熒光定量PCR和RNA-seq高通量測(cè)序分析表明,我們發(fā)現(xiàn)玉米ZmPIFs基因的表達(dá)模式存在特異性:在不同的組織部位、不同的發(fā)育時(shí)期、不同基因?qū)φ陉幒蟮捻憫?yīng);并且發(fā)現(xiàn)bHLH24和bHLH74兩個(gè)基因在短日照下的表達(dá)存在晝夜節(jié)律。(2)克隆出10個(gè)玉米ZmPIFs基因:bHLH1、bHLH12、bHLH24、bHLH30、bHLH40、bHLH44、bHLH51、bHLH113、bHLH130和bHLH180。結(jié)合基因序列的正確性、結(jié)構(gòu)域的完整性和是否在玉米中起主要作用,將其中的部分玉米ZmPIFs基因轉(zhuǎn)入到擬南芥pifq缺失突變體和野生型Col中。通過(guò)進(jìn)一步驗(yàn)證,發(fā)現(xiàn)bHLH44基因?qū)τ谝种乒庑螒B(tài)建成的作用最明顯:在黑暗環(huán)境中,bHLH44促進(jìn)了35S:bHLH44-3FLAG/pifq和35S:bHLH44-3FLAG/Col的下胚軸長(zhǎng)度,改變了35S:bHLH44-3FLAG/pifq的子葉張開(kāi)程度。其抑制光形態(tài)建成的功能類(lèi)似于A(yíng)tPIFs基因。(3)bHLH44基因影響了植株的向重力性,在黑暗環(huán)境中,缺失突變體pifq的下胚軸不存在負(fù)向地性,而35S:bHLH44-3FLAG/pifq和野生型Col的下胚軸響應(yīng)重力。我們通過(guò)I2-KI染色實(shí)驗(yàn)檢測(cè)植株體內(nèi)的淀粉合成情況解釋了轉(zhuǎn)基因幼苗可以感應(yīng)重力的原因,bHLH44基因像擬南芥AtPIF1基因一樣,阻止了植株體內(nèi)胚層造粉體向白色體的轉(zhuǎn)換。
[Abstract]:Light plays an important role in the growth and development of plants. On the one hand, it not only brings energy to plants and makes them accumulate assimilates through photosynthesis; on the other hand, light plays the role of signal molecule. Make plants adjust their growth and development through the change of induced light. Plants perceive light signals through Guang Min pigments in vivo, and regulate the growth process of Arabidopsis thaliana by the interaction of Guang Min pigments with Guang Min pigments. PIFs play an important role in Arabidopsis thaliana growth and development. Promote the elongation of hypocotyls, inhibit cotyledon opening, and inhibit photomorphogenesis. At present, the research on PIFs is mainly focused on model plants, but the research on ZmPIFs in some high crops, especially in maize, is still less. Therefore, it is of great significance to study the ZmPIFs gene family in maize. In this experiment, we identified the members of maize ZmPIFs gene family from maize gene database MaizeGDB, explored the expression pattern of the gene family, and isolated 10 maize genes. Four of the genes were transferred into Arabidopsis thaliana. The results showed that bHLH44 gene plays an important role in inhibiting light morphogenesis and affecting plant response to gravity. The main results are as follows: (1) according to the AtPIFs gene family sequence of Arabidopsis thaliana queried on NCBI, 17 ZmPIFs homologous genes of maize were obtained by BLAST analysis on MaizeGDB. The structure and function of maize ZmPIFs gene were predicted by evolutionary tree alignment, gene sequence analysis, amino acid sequence analysis and protein domain prediction. Finally, ten maize ZmPIFs genes, ZmbHLH12, ZmbHLH30, ZmbHLH37, ZmbHLH40, ZmbHLH74, ZmbHLH78, ZmbHLH83, ZmbHLH113 and ZmbHLH180, which are homologous to Arabidopsis thaliana AtPIF1, ATPIF3 and AtPIF5, have been identified, and their main roles are ZmbHLH12, ZmbHLH30, ZmbHLH40, ZmbHLH40, ZmbHLH74, ZmbHLH88, ZmbHLH113 and ZmbHLH180. Because the PIFs gene belongs to the bHLH gene family, we use its number in the bHLH gene family to name the ZmPIFs gene in maize. Real-time fluorescence quantitative PCR and RNA-seq high throughput sequencing analysis showed that the expression pattern of ZmPIFs gene in maize was specific: the response of different genes to shading was different in different tissues and different developmental stages. It was also found that there was a circadian rhythm in the expression of bHLH24 and bHLH74 genes in short sunlight. (2) Ten maize ZmPIFs genes: bHLH1, bHLH12, bHLH24, bHLH30, bHLH40, bHLH4H4BHLH113bHLH130 and bHLH180were cloned. Combined with the correctness of the gene sequence, the integrity of the domain and whether or not it plays a major role in maize, some of the maize ZmPIFs genes were transferred into Arabidopsis thaliana pifq deletion mutant and wild-type Col. Through further verification, we found that bHLH44 gene had the most obvious effect on inhibiting photomorphogenesis: in dark environment, bHLH44 promoted the length of hypocotyls of 35S:bHLH44-3FLAG/pifq and 35S:bHLH44-3FLAG/Col, and changed the opening degree of cotyledons of 35S:bHLH44-3FLAG/pifq. The inhibition of photomorphogenesis was similar to that of AtPIFs gene. (3) bHLH44 gene affected plant gravity. In dark environment, the hypocotyls of deletion mutant pifq had no negative ground, while 35S:bHLH44-3FLAG/pifq and wild-type Col Hypocotyl responded to gravity. The starch synthesis in transgenic seedlings was detected by I2-KI staining. The reason why transgenic seedlings can induce gravity is that the gene of bHLH44, like Arabidopsis AtPIF1 gene, prevents the transformation of the plantlets from endosperm to white bodies in the same way as in Arabidopsis thaliana.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：Q943.2;S513
本文編號(hào)：2181531
[Abstract]:Light plays an important role in the growth and development of plants. On the one hand, it not only brings energy to plants and makes them accumulate assimilates through photosynthesis; on the other hand, light plays the role of signal molecule. Make plants adjust their growth and development through the change of induced light. Plants perceive light signals through Guang Min pigments in vivo, and regulate the growth process of Arabidopsis thaliana by the interaction of Guang Min pigments with Guang Min pigments. PIFs play an important role in Arabidopsis thaliana growth and development. Promote the elongation of hypocotyls, inhibit cotyledon opening, and inhibit photomorphogenesis. At present, the research on PIFs is mainly focused on model plants, but the research on ZmPIFs in some high crops, especially in maize, is still less. Therefore, it is of great significance to study the ZmPIFs gene family in maize. In this experiment, we identified the members of maize ZmPIFs gene family from maize gene database MaizeGDB, explored the expression pattern of the gene family, and isolated 10 maize genes. Four of the genes were transferred into Arabidopsis thaliana. The results showed that bHLH44 gene plays an important role in inhibiting light morphogenesis and affecting plant response to gravity. The main results are as follows: (1) according to the AtPIFs gene family sequence of Arabidopsis thaliana queried on NCBI, 17 ZmPIFs homologous genes of maize were obtained by BLAST analysis on MaizeGDB. The structure and function of maize ZmPIFs gene were predicted by evolutionary tree alignment, gene sequence analysis, amino acid sequence analysis and protein domain prediction. Finally, ten maize ZmPIFs genes, ZmbHLH12, ZmbHLH30, ZmbHLH37, ZmbHLH40, ZmbHLH74, ZmbHLH78, ZmbHLH83, ZmbHLH113 and ZmbHLH180, which are homologous to Arabidopsis thaliana AtPIF1, ATPIF3 and AtPIF5, have been identified, and their main roles are ZmbHLH12, ZmbHLH30, ZmbHLH40, ZmbHLH40, ZmbHLH74, ZmbHLH88, ZmbHLH113 and ZmbHLH180. Because the PIFs gene belongs to the bHLH gene family, we use its number in the bHLH gene family to name the ZmPIFs gene in maize. Real-time fluorescence quantitative PCR and RNA-seq high throughput sequencing analysis showed that the expression pattern of ZmPIFs gene in maize was specific: the response of different genes to shading was different in different tissues and different developmental stages. It was also found that there was a circadian rhythm in the expression of bHLH24 and bHLH74 genes in short sunlight. (2) Ten maize ZmPIFs genes: bHLH1, bHLH12, bHLH24, bHLH30, bHLH40, bHLH4H4BHLH113bHLH130 and bHLH180were cloned. Combined with the correctness of the gene sequence, the integrity of the domain and whether or not it plays a major role in maize, some of the maize ZmPIFs genes were transferred into Arabidopsis thaliana pifq deletion mutant and wild-type Col. Through further verification, we found that bHLH44 gene had the most obvious effect on inhibiting photomorphogenesis: in dark environment, bHLH44 promoted the length of hypocotyls of 35S:bHLH44-3FLAG/pifq and 35S:bHLH44-3FLAG/Col, and changed the opening degree of cotyledons of 35S:bHLH44-3FLAG/pifq. The inhibition of photomorphogenesis was similar to that of AtPIFs gene. (3) bHLH44 gene affected plant gravity. In dark environment, the hypocotyls of deletion mutant pifq had no negative ground, while 35S:bHLH44-3FLAG/pifq and wild-type Col Hypocotyl responded to gravity. The starch synthesis in transgenic seedlings was detected by I2-KI staining. The reason why transgenic seedlings can induce gravity is that the gene of bHLH44, like Arabidopsis AtPIF1 gene, prevents the transformation of the plantlets from endosperm to white bodies in the same way as in Arabidopsis thaliana.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：Q943.2;S513
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