棉铃优势表达基因GhMAKR3对棉铃发育的影响
发布时间：2018-07-28 15:10
【摘要】：棉花是世界上最主要的天然纤维作物,棉花生产在我国国民经济中占有十分重要的地位。纤维产量主要由单位面积铃数、平均铃重和衣分三部分组成。产量的增加离不开各组分之间的相互协调,这三个组分均与棉铃发育密切相关。因此,研究与棉铃生长发育相关的基因,进而利用基因工程技术促进棉铃的生长发育,可为提高棉花产量和改良纤维品质提供新途径。MAKRs(Membrane Associated Kinase Regulators)蛋白是一类具有激酶调节元件的膜结合蛋白,可能参与调控不同的信号传导途径。拟南芥中AtMAKR1与AtBKI1类似,负调控BRs信号传导;AtMAKR4作用在IBA-IAA转化通路下游,促进拟南芥侧根的形成。但是,MAKRs在植物生长发育中的功能尚不清楚,相关研究报导很少,其对棉花生长发育的影响更不清楚。本研究从棉花中克隆得到一个具有棉铃优势表达特性的基因GhMAKR3。为研究GhMAKR3基因的生物学功能,对该基因在棉花中的表达模式及其上游序列的启动子特性进行了分析;构建了S7启动子调控的GhMAKR3表达上调的植物表达载体和Ca MV35S启动子调控的GhMAKR3表达下调的植物表达载体,通过农杆菌介导的棉花遗传转化获得了转基因材料;观察了上调和下调该基因表达的转基因棉花的表型。主要结果如下:1.棉花GhMAKR3基因的克隆与表达模式分析根据葡萄果实中的EST序列在棉花中克隆得到一个棉铃优势表达基因,该基因与拟南芥MAKRs家族的AtMAKR3相似性较高(95%),定名为GhMAKR3。序列分析显示,该基因全长1715 bp,包含一个长为1035 bp完整的ORF(Open Reading Frame),编码344个氨基酸残基。同源性分析显示,GhMAKR3氨基酸序列与橙子、葡萄、苹果、莲、梅花等物种的MAKRs蛋白具有较高的相似性。系统进化树分析得出,GhMAKR3与可可的未知蛋白、橙子的CsMAKR4-like、梅花的PmMAKR4、苹果的MdMAKR4有较近的亲缘关系。qRT-PCR分析GhMAKR3基因在棉花中的表达特性,结果显示该基因在胚珠、纤维、铃壳的相对表达水平较高,而在棉花的其他组织中表达水平相对较低。进一步分析GhMAKR3基因在不同时期的胚珠、纤维、铃壳中的表达,发现该基因在胚珠发育的前期(0-10 DPA,Days Post Anthesis)表达水平较高,并且在6 DPA时达到最大值;同时在纤维发育的起始期和快速伸长期(0-12 DPA)表达水平较高,并且在7 DPA时达到最大值。在铃壳发育的前期(1-4 DPA)该基因也有较高的表达水平,且在1 DPA时达到最大值。2.ghmakr3基因上游启动子调控元件分析及gus表达特性运用plantcare和place数据库分析得出,ghmakr3基因启动子序列包括多种顺式作用元件,除常见的启动区、增强区顺式作用元件外,还包含如胚乳、花粉、根等组织特异表达相关元件,生长素、赤霉素、油菜素固醇等激素响应元件,以及光、热、干旱等环境相关的响应元件。这说明ghmakr3基因的表达受到多种因素的影响。将该启动子序列与gus报告基因融合,pghmakr3::gus转基因棉花组织器官中的gus染色发现,在花瓣、雄蕊和不同发育时期的棉铃中gus活性较高,在叶片、叶柄、雌蕊部位gus活性较低。这与该基因在棉花中的表达模式基本相符。3.ghmakr3基因对brs信号传导途径相关基因表达的影响胚珠离体培养条件下,添加brz(brassinazole,br合成的特异抑制剂)棉花0dpa胚珠的ghmakr3表达水平上升,而添加bl(brassinolide,芸苔素内酯)该基因表达水平下降,这表明该基因的表达受到brs的抑制。qrt-pcr分别检测超量和抑制ghmakr3表达的转基因棉花中brs信号传导途径相关基因的表达水平。上调ghmakr3表达,brs信号传导途径上游基因ghbri1表达水平略有上升,ghbki1表达水平无明显变化;该途径下游基因ghbsu1和ghbin2表达水平上调,而ghbzr1表达水平下降。下调ghmakr3表达,ghbri1表达水平下降,ghbki1表达水平上升;而ghbsu1和ghbin2表达水平下降,ghbzr1表达水平上升。这表明ghmakr3基因表达影响brs信号传导途径相关基因的表达,可能参与brs信号传导过程。4.ghmakr3基因对纤维发育相关的myb转录因子表达的影响qrt-pcr分别检测超量和抑制ghmakr3表达的转基因棉花中与纤维发育相关的myb转录因子的表达变化。上调ghmakr3表达后ghmyb25-like、ghmyb25、ghmyb109等基因表达水平降低,且开花当天胚珠表皮纤维细胞起始密度低于同时期的野生型。下调ghmakr3表达后除ghmyb109表达水平上升外,ghmyb25-like、ghmyb25表达水平无明显变化,同时开花当天胚珠表皮纤维细胞起始密度无明显变化。5.下调ghmakr3基因可促进糖向种子的转运上调ghmakr3基因表达后,成熟种子可溶性总糖含量变化不明显,而蔗糖转运蛋白基因ghsut1-9表达水平不同程度降低;下调ghmakr3基因表达后成熟种子可溶性总糖含量较野生型提高,同时ghsut1-9表达水平有不同程度的上升。表明抑制ghmakr3的表达可以促进糖向种子的转运。6.下调ghmakr3基因促进了棉铃及种子的发育观察10dpa棉铃发现,超量表达ghmakr3基因后棉铃与苞叶均比同时期野生型变小,而干扰该基因表达后棉铃与苞叶均变大。考种和纤维检测分析得出,上调GhMAKR3基因表达棉花结铃性降低、棉铃和种子变小;而下调GhMAKR3基因表达能够促进棉铃的生长发育,使结铃数增多、棉铃和种子变大。上述结果表明,GhMAKR3基因具有棉铃优势表达特性,上调其表达抑制棉铃的生长发育;反之,干扰该基因的表达则促进棉铃的生长发育。推测GhMAKR3的表达可能与BRs水平存在负相关性,即上调该基因表达与降低BRs水平的表型类似,而抑制其表达与提高BRs水平的表型类似。
[Abstract]:Cotton is the most important natural fiber crop in the world. The production of cotton occupies a very important position in the national economy of our country. The output of fiber is mainly composed of three parts: the number of bolls per unit area, the average bell weight and the clothing score. The increase of the output can not be separated from the coordination among the components. All these three components are closely related to the development of cotton bolls. Therefore, Study the genes related to the growth and development of cotton boll, and then use gene engineering technology to promote the growth and development of cotton boll, which can provide a new way to improve the cotton yield and improve the quality of fiber.MAKRs (Membrane Associated Kinase Regulators) protein is a kind of membrane binding protein with kinase regulator element, which may participate in the regulation of different signals. AtMAKR1 is similar to AtBKI1 in Arabidopsis, which negatively regulates BRs signal conduction; AtMAKR4 plays a role in the formation of lateral roots of Arabidopsis in the downstream of IBA-IAA transformation pathway. However, the function of MAKRs in plant growth is not clear, and the related reports are few, and the effect of this study is less clear on the growth and development of cotton. A gene GhMAKR3. with the advantage of cotton boll expression was obtained to study the biological function of the GhMAKR3 gene. The expression pattern of the gene in cotton and the promoter characteristics of the upstream sequence were analyzed. The plant expression vector regulated by the S7 promoter and the GhMAKR3 regulated by the Ca MV35S promoter were constructed. The expression vector of down regulated plant was expressed and transgenic material was obtained through genetic transformation of Agrobacterium tumefaciens, and the phenotype of transgenic cotton was up-regulated and down regulated. The main results were as follows: 1. the cloning and expression pattern analysis of GhMAKR3 gene of cotton was cloned in cotton by EST sequence in grape fruit. The gene of cotton boll dominant expression was similar to that of AtMAKR3 in MAKRs family of Arabidopsis (95%). It was named GhMAKR3. sequence analysis showing that the gene was 1715 BP, including a 1035 BP complete ORF (Open Reading Frame) and 344 amino acid residues. Homology analysis showed that the GhMAKR3 amino acid sequence was with oranges, grapes, The MAKRs protein of apple, lotus, plum and other species has high similarity. Phylogenetic tree analysis showed that GhMAKR3 was closely related to the unknown protein of cocoa, CsMAKR4-like of orange, PmMAKR4 of plum blossom, and MdMAKR4 of apple,.QRT-PCR analysis of the expression of GhMAKR3 gene in cotton. The result showed that the gene was in ovule and fiber, and the result showed that the gene was in ovule and fiber, The relative expression level of the bell shell was relatively high and the expression level was relatively low in other tissues of cotton. The expression of GhMAKR3 gene in the ovules, fibers and bell shells at different stages was further analyzed. It was found that the gene reached a higher level in the early stage of the ovule development (0-10 DPA, Days Post Anthesis) and reached the maximum at 6 DPA; at the same time, the gene was at the same time. The expression level of the initial and rapid elongation period (0-12 DPA) of fiber development is higher and reaches the maximum at 7 DPA. In the early stage of the bell shell development (1-4 DPA), the gene also has a higher expression level, and at 1 DPA, the maximum value of the upstream promoter of.2.ghmakr3 gene is analyzed and the gus expression characteristics use plantcare and place data. The library analysis shows that the ghmakr3 gene promoter sequence includes a variety of cis acting elements. Besides the common starting areas and the cis acting elements in the enhanced region, it also contains response elements such as endosperm, pollen, root and other tissue specific expression components, auxin, gibberellin, brassin and sterols, as well as response elements related to light, heat, and drought. This indicates that the expression of ghmakr3 gene is affected by a variety of factors. Fusion of the promoter sequence and the gus reporter gene. Gus staining in pghmakr3:: Gus transgenic cotton tissues and organs found that the GUS activity in the petals, stamens and Cotton Bolls at different developmental stages is higher in the leaves, petioles, and pistil sites, and this is in cotton with the gene in cotton. The expression pattern in the flower basically conforms to the effect of the.3.ghmakr3 gene on the expression of BRS signaling pathway related genes, and the expression level of ghmakr3 in cotton 0dpa ovules increases with the addition of brz (brassinazole, Br synthesis inhibitor), and the expression level of the gene is decreased by adding BL (brassinolide, brassinolide). The expression of the gene was inhibited by BRS inhibition.Qrt-pcr to detect the expression level of BRS signal transduction pathway related genes in transgenic cotton with overexpression and inhibition of ghmakr3 expression. The expression of ghmakr3 was up regulated, the expression level of ghbri1 in the upstream gene of BRS signal transduction pathway increased slightly, and the level of ghbki1 table reached no obvious change; the downstream gene GHB in this way was GHB. The expression level of SU1 and ghbin2 was up, while the expression level of ghbzr1 decreased. The expression level of ghmakr3 decreased, the expression level of ghbri1 decreased, and the expression level of ghbki1 increased, while the expression level of ghbsu1 and ghbin2 decreased, and the ghbzr1 expression level increased. This indicates that the expression of ghmakr3 gene affects the expression of the genes related to the conduction path of BRS signal, and may be involved in the conduction of BRS signals. The effect of.4.ghmakr3 gene on the expression of MYB transcription factors related to fiber development, qRT-PCR detected the changes in the expression of MYB transcriptional factors related to fiber development in transgenic cotton with excess and inhibition of ghmakr3 expression. The expression level of ghmyb25-like, ghmyb25, ghmyb109 and other gene expression levels decreased after up regulation of ghmakr3 expression, and the ovule table on the day of flowering The initial density of the skin fibroblasts was lower than that of the wild type. The expression level of ghmyb25-like and ghmyb25 was not obviously changed, except the expression level of ghmyb109, and there was no obvious change in the initial density of the epidermal fibroblast in the ovule on the same day, and the.5. down regulation of ghmakr3 gene could promote the up-regulation of the ghmakr3 gene of sugar to the seed by the decrease of the expression level of ghmakr3. After the expression, the content of soluble total sugar in mature seeds was not obvious, but the expression level of ghsut1-9 in the sucrose transporter gene decreased in different degrees, and the content of soluble total sugar in mature seeds was higher than that of wild type after down-regulation of ghmakr3 gene expression, while the expression level of ghsut1-9 increased in varying degrees. It indicated that the inhibition of ghmakr3 expression could promote sugar. The down-regulation of ghmakr3 gene to the seed transport.6. promoted the development of cotton bolls and seeds to observe the discovery of 10dpa cotton bolls. After the overexpression of ghmakr3 gene, both cotton bolls and bracts were smaller than those of the same period wild type, and the cotton bolls and bracts became larger after interference of the gene expression. Test and fiber detection analysis showed that the GhMAKR3 gene was raised to express the boll property of cotton. The reduction of cotton bolls and seeds decreased, while down regulation of GhMAKR3 gene expression could promote the growth and development of cotton bolls, increase the number of bolls, and increase the cotton bolls and seeds. The results showed that the GhMAKR3 gene had the advantage of cotton boll expression, up regulation of its expression to inhibit the growth and development of cotton bolls, and the expression of the gene could promote the growth and development of cotton bolls. It is presumed that the expression of GhMAKR3 may have a negative correlation with the level of BRs, that is, up - regulation of the gene expression and the phenotype of the BRs level, while inhibiting the expression of the gene is similar to the phenotype that increases the level of BRs.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S562
本文编号：2150647
[Abstract]:Cotton is the most important natural fiber crop in the world. The production of cotton occupies a very important position in the national economy of our country. The output of fiber is mainly composed of three parts: the number of bolls per unit area, the average bell weight and the clothing score. The increase of the output can not be separated from the coordination among the components. All these three components are closely related to the development of cotton bolls. Therefore, Study the genes related to the growth and development of cotton boll, and then use gene engineering technology to promote the growth and development of cotton boll, which can provide a new way to improve the cotton yield and improve the quality of fiber.MAKRs (Membrane Associated Kinase Regulators) protein is a kind of membrane binding protein with kinase regulator element, which may participate in the regulation of different signals. AtMAKR1 is similar to AtBKI1 in Arabidopsis, which negatively regulates BRs signal conduction; AtMAKR4 plays a role in the formation of lateral roots of Arabidopsis in the downstream of IBA-IAA transformation pathway. However, the function of MAKRs in plant growth is not clear, and the related reports are few, and the effect of this study is less clear on the growth and development of cotton. A gene GhMAKR3. with the advantage of cotton boll expression was obtained to study the biological function of the GhMAKR3 gene. The expression pattern of the gene in cotton and the promoter characteristics of the upstream sequence were analyzed. The plant expression vector regulated by the S7 promoter and the GhMAKR3 regulated by the Ca MV35S promoter were constructed. The expression vector of down regulated plant was expressed and transgenic material was obtained through genetic transformation of Agrobacterium tumefaciens, and the phenotype of transgenic cotton was up-regulated and down regulated. The main results were as follows: 1. the cloning and expression pattern analysis of GhMAKR3 gene of cotton was cloned in cotton by EST sequence in grape fruit. The gene of cotton boll dominant expression was similar to that of AtMAKR3 in MAKRs family of Arabidopsis (95%). It was named GhMAKR3. sequence analysis showing that the gene was 1715 BP, including a 1035 BP complete ORF (Open Reading Frame) and 344 amino acid residues. Homology analysis showed that the GhMAKR3 amino acid sequence was with oranges, grapes, The MAKRs protein of apple, lotus, plum and other species has high similarity. Phylogenetic tree analysis showed that GhMAKR3 was closely related to the unknown protein of cocoa, CsMAKR4-like of orange, PmMAKR4 of plum blossom, and MdMAKR4 of apple,.QRT-PCR analysis of the expression of GhMAKR3 gene in cotton. The result showed that the gene was in ovule and fiber, and the result showed that the gene was in ovule and fiber, The relative expression level of the bell shell was relatively high and the expression level was relatively low in other tissues of cotton. The expression of GhMAKR3 gene in the ovules, fibers and bell shells at different stages was further analyzed. It was found that the gene reached a higher level in the early stage of the ovule development (0-10 DPA, Days Post Anthesis) and reached the maximum at 6 DPA; at the same time, the gene was at the same time. The expression level of the initial and rapid elongation period (0-12 DPA) of fiber development is higher and reaches the maximum at 7 DPA. In the early stage of the bell shell development (1-4 DPA), the gene also has a higher expression level, and at 1 DPA, the maximum value of the upstream promoter of.2.ghmakr3 gene is analyzed and the gus expression characteristics use plantcare and place data. The library analysis shows that the ghmakr3 gene promoter sequence includes a variety of cis acting elements. Besides the common starting areas and the cis acting elements in the enhanced region, it also contains response elements such as endosperm, pollen, root and other tissue specific expression components, auxin, gibberellin, brassin and sterols, as well as response elements related to light, heat, and drought. This indicates that the expression of ghmakr3 gene is affected by a variety of factors. Fusion of the promoter sequence and the gus reporter gene. Gus staining in pghmakr3:: Gus transgenic cotton tissues and organs found that the GUS activity in the petals, stamens and Cotton Bolls at different developmental stages is higher in the leaves, petioles, and pistil sites, and this is in cotton with the gene in cotton. The expression pattern in the flower basically conforms to the effect of the.3.ghmakr3 gene on the expression of BRS signaling pathway related genes, and the expression level of ghmakr3 in cotton 0dpa ovules increases with the addition of brz (brassinazole, Br synthesis inhibitor), and the expression level of the gene is decreased by adding BL (brassinolide, brassinolide). The expression of the gene was inhibited by BRS inhibition.Qrt-pcr to detect the expression level of BRS signal transduction pathway related genes in transgenic cotton with overexpression and inhibition of ghmakr3 expression. The expression of ghmakr3 was up regulated, the expression level of ghbri1 in the upstream gene of BRS signal transduction pathway increased slightly, and the level of ghbki1 table reached no obvious change; the downstream gene GHB in this way was GHB. The expression level of SU1 and ghbin2 was up, while the expression level of ghbzr1 decreased. The expression level of ghmakr3 decreased, the expression level of ghbri1 decreased, and the expression level of ghbki1 increased, while the expression level of ghbsu1 and ghbin2 decreased, and the ghbzr1 expression level increased. This indicates that the expression of ghmakr3 gene affects the expression of the genes related to the conduction path of BRS signal, and may be involved in the conduction of BRS signals. The effect of.4.ghmakr3 gene on the expression of MYB transcription factors related to fiber development, qRT-PCR detected the changes in the expression of MYB transcriptional factors related to fiber development in transgenic cotton with excess and inhibition of ghmakr3 expression. The expression level of ghmyb25-like, ghmyb25, ghmyb109 and other gene expression levels decreased after up regulation of ghmakr3 expression, and the ovule table on the day of flowering The initial density of the skin fibroblasts was lower than that of the wild type. The expression level of ghmyb25-like and ghmyb25 was not obviously changed, except the expression level of ghmyb109, and there was no obvious change in the initial density of the epidermal fibroblast in the ovule on the same day, and the.5. down regulation of ghmakr3 gene could promote the up-regulation of the ghmakr3 gene of sugar to the seed by the decrease of the expression level of ghmakr3. After the expression, the content of soluble total sugar in mature seeds was not obvious, but the expression level of ghsut1-9 in the sucrose transporter gene decreased in different degrees, and the content of soluble total sugar in mature seeds was higher than that of wild type after down-regulation of ghmakr3 gene expression, while the expression level of ghsut1-9 increased in varying degrees. It indicated that the inhibition of ghmakr3 expression could promote sugar. The down-regulation of ghmakr3 gene to the seed transport.6. promoted the development of cotton bolls and seeds to observe the discovery of 10dpa cotton bolls. After the overexpression of ghmakr3 gene, both cotton bolls and bracts were smaller than those of the same period wild type, and the cotton bolls and bracts became larger after interference of the gene expression. Test and fiber detection analysis showed that the GhMAKR3 gene was raised to express the boll property of cotton. The reduction of cotton bolls and seeds decreased, while down regulation of GhMAKR3 gene expression could promote the growth and development of cotton bolls, increase the number of bolls, and increase the cotton bolls and seeds. The results showed that the GhMAKR3 gene had the advantage of cotton boll expression, up regulation of its expression to inhibit the growth and development of cotton bolls, and the expression of the gene could promote the growth and development of cotton bolls. It is presumed that the expression of GhMAKR3 may have a negative correlation with the level of BRs, that is, up - regulation of the gene expression and the phenotype of the BRs level, while inhibiting the expression of the gene is similar to the phenotype that increases the level of BRs.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S562
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1 刘通;棉铃优势表达基因GhMAKR3对棉铃发育的影响[D];西南大学;2016年
，本文编号：2150647
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