生長素響應因子GhARF6對棉花生長發(fā)育的影響
發(fā)布時間：2019-05-30 03:37
【摘要】：棉花是關系國計民生的重要物資,在國民經(jīng)濟發(fā)展中占有重要的地位。生長素可以調(diào)控細胞的伸長、分裂和分化,不定根的形成、果實的發(fā)育和胚的形成等。生長素通過其信號途徑對功能基因進行調(diào)控,從而影響植物的生長發(fā)育。因此,生長素信號傳導途徑及其對下游基因的調(diào)控機理是近年來植物生物學研究的熱點。棉花中生長素信號傳導相關基因的生物學功能進行研究,有助于解生長素在棉花生長發(fā)育以及形態(tài)建成過程中所發(fā)揮的作用,在闡明棉花中生長素信號傳導的復雜分子機制方面具有重要的理論意義。ARFs(Auxin response factors)基因家族的發(fā)現(xiàn),使人們對生長素信號傳導途徑的認識有了突破性的進展。ARF6基因與植物頂端分生組織的生長、維管組織的發(fā)育、解除頂芽休眠和花器官的發(fā)育相關。為了進一步解析ARF6基因在陸地棉發(fā)育過程中的功能,本論文首先克隆了GhARF6基因,分析其啟動子的表達特性。通過調(diào)控GhARF6基因在棉花中的表達水平,來探究GhARF6基因?qū)γ藁ㄉL發(fā)育的影響。具體研究結(jié)果如下:1.GhARF6基因的克隆與表達特性分析首先從陸地棉中克隆了ARF6基因的同源基因GhARF6基因。該基因位于26條染色體中的5號染色體上,其含有一個2715 bp的ORF框,編碼904個氨基酸殘基,蛋白分子量為99.78 kDa,p I=6.33。擬南芥中ARFs被分為5類,將檢索到的96個陸地棉ARF基因與擬南芥ARF基因各個分類中具有代表性的10個基因氨基酸序列進行進化樹分析顯示:陸地棉中含有8個ARF亞家族,與GhARF6基因相似度高的GhARF6-1和GhARF6-2與AtARF6、AtARF8分在了同一個亞家族,且與AtARF6基因同源性較高。對GhARF6基因氨基酸序列結(jié)構分析顯示其具有典型的ARF蛋白結(jié)構,并且在440-620氨基酸之間富集了谷氨酰胺(Q),推測其為轉(zhuǎn)錄激活子。在棉花中進行表達分析發(fā)現(xiàn)GhARF6基因在棉花植株的各個部位均有表達,莖中表達水平相對最高,是葉片中表達水平的10.1倍。在纖維和胚珠生長發(fā)育過程中表達分析顯示該基因在0-4 DPA的纖維中相對表達水平較高,0-6 DPA的胚珠中相對于其他階段表達水平較高,并在開花后6天的胚珠中達到頂峰。預示該基因在胚珠發(fā)育起始階段發(fā)揮重要作用。用不同激素對棉花幼苗進行處理,結(jié)果顯示:GhARF6基因在子葉、下胚軸和根部均有表達。GhARF6基因在IAA、NPA、NAA處理的子葉中表達水平相對較高,相對表達水平分別是未處理的12.7倍、11.5倍、9.0倍;用IAA、NAA、NPA處理后下胚軸中GhARF6基因的相對表達水平是未處理的8.3倍、3.4倍、4.2倍;在根部的表達水平在處理前后無明顯變化。用不同濃度的Al3+處理棉花幼苗發(fā)現(xiàn):0?M、25?M、50?M Al3+濃度下GhARF6基因隨著Al3+濃度的增加表達量增加,Al3+濃度為50?M時GhARF6基因表達水平達到最高。50?M、100?M、200?M Al3+濃度下GhARF6基因的表達水平隨著Al3+濃度的增加而下降。將棉花幼苗在50?M Al3+的培養(yǎng)液中進行了不同時間段的處理,發(fā)現(xiàn)GhARF6基因的表達量在0-3 h內(nèi)上調(diào),3 h后開始下降,9 h后表達水平恢復到未處理前的水平。2.GhARF6基因啟動子克隆及表達特性分析為了進一步了解GhARF6基因的功能,課題組前期克隆了該基因上游2100 bp的啟動子序列,經(jīng)PlantCARE網(wǎng)站分析發(fā)現(xiàn)該序列含有眾多的植物順式元件,包含生長素響應元件、光響應相關元件、組織表達相關元件、水楊酸響應元件、防御及脅迫響應的相關元件、特定部位表達相關的元件等。將該啟動子與GUS基因融合后分別轉(zhuǎn)化棉花和擬南芥,組織化學分析發(fā)現(xiàn):擬南芥中該啟動子主要在轉(zhuǎn)基因植株的蓮座葉、角果、花柄、花絲、柱頭頂部、主根、側(cè)根和根毛中表達。其中在花柄、主根和側(cè)根中表達活性相對較強,GUS基因在轉(zhuǎn)基因棉花植株的各個部位均有表達,主要集中在莖、下胚軸、根的維管束組織中。該基因啟動子在擬南芥與棉花中的表達模式基本相一致。對PGhARF6轉(zhuǎn)基因擬南芥用Al3+處理后發(fā)現(xiàn):用50?M Al3+處理9 h后擬南芥的根尖、下胚軸、子葉相對未處理著色深。3.調(diào)控GhARF6基因影響棉花生長發(fā)育為了進一步探究陸地棉中GhARF6基因的生物學功能,構建CaMV35S組成型啟動子控制的GhARF6基因反義抑制表達載體、超量表達載體、RNA干擾表達載體,并分別對棉花進行遺傳轉(zhuǎn)化。對轉(zhuǎn)基因棉花植株進行表型觀察發(fā)現(xiàn):下調(diào)GhARF6基因后,棉花花器官呈現(xiàn)特異表型:花瓣小,雄蕊短且花藥延遲釋放。同時伴隨著落鈴的現(xiàn)象,RNA干擾植株落鈴較為嚴重,有的達到了100%。上調(diào)GhARF6基因后,棉花花器官發(fā)育無明顯變化。下調(diào)GhARF6基因后,種子變得不飽滿并且單株單個棉鈴中種子明顯減少:反義轉(zhuǎn)基因植株3-4-3、41-1-1和干擾轉(zhuǎn)基因植株2-2、2-4分別減少了42%、33.5%、39.6%、29.7%;對轉(zhuǎn)基因棉花植株單株成熟纖維的長度測量發(fā)現(xiàn):成熟纖維變短,這種表型在RNAi轉(zhuǎn)基因植株中更為明顯。與野生型相比,反義轉(zhuǎn)基因植株3-4-3、41-1-1和干擾轉(zhuǎn)基因植株2-2、2-4轉(zhuǎn)基因棉花成熟棉纖維長度分別減少了12%、14.5%、15.4%、19.3%。然而,上調(diào)GhARF6基因?qū)γ藁ǚN子和纖維的生長發(fā)育無明顯影響。
[Abstract]:The cotton is an important material for the national economy and the people's livelihood, and plays an important role in the development of the national economy. The auxin can regulate the elongation, division and differentiation of the cell, the formation of the adventitious root, the development of the fruit, the formation of the embryo, and the like. The auxin regulates the function gene through its signal pathway, thus affecting the growth and development of the plant. Therefore, the pathway of auxin signaling and the regulation mechanism of the downstream gene are the hot spots in the research of plant biology in recent years. The study on the biological function of auxin signaling related genes in cotton is of great theoretical significance in elucidating the complex molecular mechanism of auxin signaling in cotton. The discovery of the ARFs gene family has made a breakthrough in the understanding of the pathway of auxin signaling. The growth of the ARF6 gene and the apical meristem of the plant, the development of vascular tissue, the release of the dormancy of the apical bud and the development of the flower organ. In order to further analyze the function of the ARF6 gene in the development of the upland cotton, the GhARF6 gene was first cloned and the expression of the promoter was analyzed. The effects of the GhARF6 gene on the growth and development of cotton were investigated by regulating the expression level of the GhARF6 gene in the cotton. The specific results are as follows:1. The cloning and expression of the GhARF6 gene is the first to clone the gene GhARF6 of the ARF6 gene from the upland cotton. The gene is located on chromosome 5 of the 26 chromosomes, which contains a 2715 bp ORF frame encoding 904 amino acid residues with a protein molecular weight of 99.78 kDa and p I = 6.33. The ARFs in Arabidopsis were divided into 5 classes, and the retrieved 96 upland cotton ARF genes were compared with the representative 10 gene amino acid sequences in each classification of the Arabidopsis ARF gene to show that the upland cotton contains 8 ARF subfamilies, GhARF6-1 and GhARF6-2 and AARF6 with high similarity to the GhARF6 gene, AtARF8 is in the same subfamily and has a high homology to the AARF6 gene. The structural analysis of the amino acid sequence of the GhARF6 gene shows that it has a typical ARF protein structure and is enriched in the 440-620 amino acid with a glutaamine (Q), which is presumed to be a transcriptional activator. The expression of the GhARF6 gene in various parts of the cotton plant was found in the cotton, and the expression level of the stem was relatively high, which was 10.1 times that of the expression level in the leaves. The expression of the gene in the growth and development of the fiber and the ovule showed that the relative expression level of the gene in the fiber of 0-4DPA was high, the expression level of the ovule of 0-6DPA was high relative to the other stage, and the peak was reached in the ovule of 6 days after flowering. It indicates that the gene plays an important role in the initiation stage of the development of the ovule. The results showed that the GhARF6 gene was expressed in the cotyledons, hypocotyl and root. The expression level of the GhARF6 gene in the cotyledons treated with IAA, NPA and NAA was 12.7-fold, 11.5-fold and 9.0-fold, respectively. The relative expression level of the GhARF6 gene in hypocotyl was 8.3-fold, 3.4-fold and 4.2-fold with IAA, NAA and NPA. The expression level of the roots did not change significantly before and after treatment. The expression level of GhARF6 gene increased with the increase of Al3 + concentration and the expression level of GhARF6 was the highest when the concentration of Al3 + was 50? M, with the increase of Al3 + concentration in the cotton seedlings treated with Al3 + treated with different concentration. The expression level of GhARF6 gene in the concentration of 200? M Al3 + decreased with the increase of Al3 + concentration. the cotton seedlings are treated in a culture solution of 50-M Al3 + for different time periods, The expression of the GhARF6 gene was up-regulated within 0-3 h, decreased after 3 h, and the expression level after 9 h was restored to the pre-treated level. The PlantCARRE site analysis found that the sequence contained numerous plant cis-elements, including auxin response elements, light response related elements, tissue expression related elements, salicylic acid response elements, defence and stress response related elements, specific site expression related elements, and the like. After the promoter and the GUS gene were fused, the cotton and the Arabidopsis were respectively transformed, and the chemical analysis of the tissue found that the promoter is mainly expressed in the lotus leaf, the corner fruit, the flower stem, the flower filament, the head of the stigma, the main root, the lateral root and the root hairs of the transgenic plant. Wherein the expression activity of the GUS gene in the stem, the main root and the lateral root is relatively strong, and the GUS gene is expressed in various parts of the transgenic cotton plant, and is mainly concentrated in the vascular bundle tissues of the stem, the hypocotyl and the root. The gene promoter is basically consistent with the expression pattern of the Arabidopsis and the cotton. It was found that the root tip, hypocotyl and cotyledons of Arabidopsis were treated with 50? M Al3 + for 9 h. In ord to further study that biological function of the GhARF6 gene in the Gossypium hirsutum L., the GhARF6 gene antisense expression vector, the over-expression vector and the RNA interference expression vector control by the CaMV35S constitutive promoter are constructed, and the cotton is respectively genetically transformed. Phenotypic observation of transgenic cotton plants showed that, after the GhARF6 gene was down-regulated, the cotton flower organs exhibited a specific phenotype: the petals were small, the stamens were short, and the anthers were delayed release. At the same time, with the phenomenon of the landing bell, the ring of the RNA interference plant is more serious and has reached 100%. After up-regulation of the GhARF6 gene, there was no significant change in the development of the floral organ of the cotton. After the GhARF6 gene was down-regulated, the seed became less plump and the seed of single boll in single plant was significantly reduced:3-4-3,41-1-1 and 2-2,2-4 of the antisense transgenic plants were reduced by 42%, 33.5%, 39.6% and 29.7%, respectively; The mature fiber becomes shorter, and the phenotype is more obvious in the RNAi transgenic plant. Compared with the wild type, the length of the mature cotton fiber of the transgenic cotton 3-4-3,41-1-1 and the transgenic plants 2-2,2-4 decreased by 12%, 14.5%, 15.4% and 19.3%, respectively. However, the up-regulation of the GhARF6 gene has no significant effect on the growth and development of cotton seeds and fibers.
【學位授予單位】：西南大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S562
本文編號：2488488
[Abstract]:The cotton is an important material for the national economy and the people's livelihood, and plays an important role in the development of the national economy. The auxin can regulate the elongation, division and differentiation of the cell, the formation of the adventitious root, the development of the fruit, the formation of the embryo, and the like. The auxin regulates the function gene through its signal pathway, thus affecting the growth and development of the plant. Therefore, the pathway of auxin signaling and the regulation mechanism of the downstream gene are the hot spots in the research of plant biology in recent years. The study on the biological function of auxin signaling related genes in cotton is of great theoretical significance in elucidating the complex molecular mechanism of auxin signaling in cotton. The discovery of the ARFs gene family has made a breakthrough in the understanding of the pathway of auxin signaling. The growth of the ARF6 gene and the apical meristem of the plant, the development of vascular tissue, the release of the dormancy of the apical bud and the development of the flower organ. In order to further analyze the function of the ARF6 gene in the development of the upland cotton, the GhARF6 gene was first cloned and the expression of the promoter was analyzed. The effects of the GhARF6 gene on the growth and development of cotton were investigated by regulating the expression level of the GhARF6 gene in the cotton. The specific results are as follows:1. The cloning and expression of the GhARF6 gene is the first to clone the gene GhARF6 of the ARF6 gene from the upland cotton. The gene is located on chromosome 5 of the 26 chromosomes, which contains a 2715 bp ORF frame encoding 904 amino acid residues with a protein molecular weight of 99.78 kDa and p I = 6.33. The ARFs in Arabidopsis were divided into 5 classes, and the retrieved 96 upland cotton ARF genes were compared with the representative 10 gene amino acid sequences in each classification of the Arabidopsis ARF gene to show that the upland cotton contains 8 ARF subfamilies, GhARF6-1 and GhARF6-2 and AARF6 with high similarity to the GhARF6 gene, AtARF8 is in the same subfamily and has a high homology to the AARF6 gene. The structural analysis of the amino acid sequence of the GhARF6 gene shows that it has a typical ARF protein structure and is enriched in the 440-620 amino acid with a glutaamine (Q), which is presumed to be a transcriptional activator. The expression of the GhARF6 gene in various parts of the cotton plant was found in the cotton, and the expression level of the stem was relatively high, which was 10.1 times that of the expression level in the leaves. The expression of the gene in the growth and development of the fiber and the ovule showed that the relative expression level of the gene in the fiber of 0-4DPA was high, the expression level of the ovule of 0-6DPA was high relative to the other stage, and the peak was reached in the ovule of 6 days after flowering. It indicates that the gene plays an important role in the initiation stage of the development of the ovule. The results showed that the GhARF6 gene was expressed in the cotyledons, hypocotyl and root. The expression level of the GhARF6 gene in the cotyledons treated with IAA, NPA and NAA was 12.7-fold, 11.5-fold and 9.0-fold, respectively. The relative expression level of the GhARF6 gene in hypocotyl was 8.3-fold, 3.4-fold and 4.2-fold with IAA, NAA and NPA. The expression level of the roots did not change significantly before and after treatment. The expression level of GhARF6 gene increased with the increase of Al3 + concentration and the expression level of GhARF6 was the highest when the concentration of Al3 + was 50? M, with the increase of Al3 + concentration in the cotton seedlings treated with Al3 + treated with different concentration. The expression level of GhARF6 gene in the concentration of 200? M Al3 + decreased with the increase of Al3 + concentration. the cotton seedlings are treated in a culture solution of 50-M Al3 + for different time periods, The expression of the GhARF6 gene was up-regulated within 0-3 h, decreased after 3 h, and the expression level after 9 h was restored to the pre-treated level. The PlantCARRE site analysis found that the sequence contained numerous plant cis-elements, including auxin response elements, light response related elements, tissue expression related elements, salicylic acid response elements, defence and stress response related elements, specific site expression related elements, and the like. After the promoter and the GUS gene were fused, the cotton and the Arabidopsis were respectively transformed, and the chemical analysis of the tissue found that the promoter is mainly expressed in the lotus leaf, the corner fruit, the flower stem, the flower filament, the head of the stigma, the main root, the lateral root and the root hairs of the transgenic plant. Wherein the expression activity of the GUS gene in the stem, the main root and the lateral root is relatively strong, and the GUS gene is expressed in various parts of the transgenic cotton plant, and is mainly concentrated in the vascular bundle tissues of the stem, the hypocotyl and the root. The gene promoter is basically consistent with the expression pattern of the Arabidopsis and the cotton. It was found that the root tip, hypocotyl and cotyledons of Arabidopsis were treated with 50? M Al3 + for 9 h. In ord to further study that biological function of the GhARF6 gene in the Gossypium hirsutum L., the GhARF6 gene antisense expression vector, the over-expression vector and the RNA interference expression vector control by the CaMV35S constitutive promoter are constructed, and the cotton is respectively genetically transformed. Phenotypic observation of transgenic cotton plants showed that, after the GhARF6 gene was down-regulated, the cotton flower organs exhibited a specific phenotype: the petals were small, the stamens were short, and the anthers were delayed release. At the same time, with the phenomenon of the landing bell, the ring of the RNA interference plant is more serious and has reached 100%. After up-regulation of the GhARF6 gene, there was no significant change in the development of the floral organ of the cotton. After the GhARF6 gene was down-regulated, the seed became less plump and the seed of single boll in single plant was significantly reduced:3-4-3,41-1-1 and 2-2,2-4 of the antisense transgenic plants were reduced by 42%, 33.5%, 39.6% and 29.7%, respectively; The mature fiber becomes shorter, and the phenotype is more obvious in the RNAi transgenic plant. Compared with the wild type, the length of the mature cotton fiber of the transgenic cotton 3-4-3,41-1-1 and the transgenic plants 2-2,2-4 decreased by 12%, 14.5%, 15.4% and 19.3%, respectively. However, the up-regulation of the GhARF6 gene has no significant effect on the growth and development of cotton seeds and fibers.
【學位授予單位】：西南大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S562
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