【摘要】：棉花是世界上最主要的天然纖維作物,棉花生產(chǎn)在我國國民經(jīng)濟中占有十分重要的地位。纖維產(chǎn)量主要由單位面積鈴數(shù)、平均鈴重和衣分三部分組成。產(chǎn)量的增加離不開各組分之間的相互協(xié)調(diào),這三個組分均與棉鈴發(fā)育密切相關(guān)。因此,研究與棉鈴生長發(fā)育相關(guān)的基因,進而利用基因工程技術(shù)促進棉鈴的生長發(fā)育,可為提高棉花產(chǎn)量和改良纖維品質(zhì)提供新途徑。MAKRs(Membrane Associated Kinase Regulators)蛋白是一類具有激酶調(diào)節(jié)元件的膜結(jié)合蛋白,可能參與調(diào)控不同的信號傳導(dǎo)途徑。擬南芥中AtMAKR1與AtBKI1類似,負調(diào)控BRs信號傳導(dǎo);AtMAKR4作用在IBA-IAA轉(zhuǎn)化通路下游,促進擬南芥?zhèn)雀男纬�。但�?MAKRs在植物生長發(fā)育中的功能尚不清楚,相關(guān)研究報導(dǎo)很少,其對棉花生長發(fā)育的影響更不清楚。本研究從棉花中克隆得到一個具有棉鈴優(yōu)勢表達特性的基因GhMAKR3。為研究GhMAKR3基因的生物學(xué)功能,對該基因在棉花中的表達模式及其上游序列的啟動子特性進行了分析;構(gòu)建了S7啟動子調(diào)控的GhMAKR3表達上調(diào)的植物表達載體和Ca MV35S啟動子調(diào)控的GhMAKR3表達下調(diào)的植物表達載體,通過農(nóng)桿菌介導(dǎo)的棉花遺傳轉(zhuǎn)化獲得了轉(zhuǎn)基因材料;觀察了上調(diào)和下調(diào)該基因表達的轉(zhuǎn)基因棉花的表型。主要結(jié)果如下:1.棉花GhMAKR3基因的克隆與表達模式分析根據(jù)葡萄果實中的EST序列在棉花中克隆得到一個棉鈴優(yōu)勢表達基因,該基因與擬南芥MAKRs家族的AtMAKR3相似性較高(95%),定名為GhMAKR3。序列分析顯示,該基因全長1715 bp,包含一個長為1035 bp完整的ORF(Open Reading Frame),編碼344個氨基酸殘基。同源性分析顯示,GhMAKR3氨基酸序列與橙子、葡萄、蘋果、蓮、梅花等物種的MAKRs蛋白具有較高的相似性。系統(tǒng)進化樹分析得出,GhMAKR3與可可的未知蛋白、橙子的CsMAKR4-like、梅花的PmMAKR4、蘋果的MdMAKR4有較近的親緣關(guān)系。qRT-PCR分析GhMAKR3基因在棉花中的表達特性,結(jié)果顯示該基因在胚珠、纖維、鈴殼的相對表達水平較高,而在棉花的其他組織中表達水平相對較低。進一步分析GhMAKR3基因在不同時期的胚珠、纖維、鈴殼中的表達,發(fā)現(xiàn)該基因在胚珠發(fā)育的前期(0-10 DPA,Days Post Anthesis)表達水平較高,并且在6 DPA時達到最大值;同時在纖維發(fā)育的起始期和快速伸長期(0-12 DPA)表達水平較高,并且在7 DPA時達到最大值。在鈴殼發(fā)育的前期(1-4 DPA)該基因也有較高的表達水平,且在1 DPA時達到最大值。2.ghmakr3基因上游啟動子調(diào)控元件分析及gus表達特性運用plantcare和place數(shù)據(jù)庫分析得出,ghmakr3基因啟動子序列包括多種順式作用元件,除常見的啟動區(qū)、增強區(qū)順式作用元件外,還包含如胚乳、花粉、根等組織特異表達相關(guān)元件,生長素、赤霉素、油菜素固醇等激素響應(yīng)元件,以及光、熱、干旱等環(huán)境相關(guān)的響應(yīng)元件。這說明ghmakr3基因的表達受到多種因素的影響。將該啟動子序列與gus報告基因融合,pghmakr3::gus轉(zhuǎn)基因棉花組織器官中的gus染色發(fā)現(xiàn),在花瓣、雄蕊和不同發(fā)育時期的棉鈴中g(shù)us活性較高,在葉片、葉柄、雌蕊部位gus活性較低。這與該基因在棉花中的表達模式基本相符。3.ghmakr3基因?qū)rs信號傳導(dǎo)途徑相關(guān)基因表達的影響胚珠離體培養(yǎng)條件下,添加brz(brassinazole,br合成的特異抑制劑)棉花0dpa胚珠的ghmakr3表達水平上升,而添加bl(brassinolide,蕓苔素內(nèi)酯)該基因表達水平下降,這表明該基因的表達受到brs的抑制。qrt-pcr分別檢測超量和抑制ghmakr3表達的轉(zhuǎn)基因棉花中brs信號傳導(dǎo)途徑相關(guān)基因的表達水平。上調(diào)ghmakr3表達,brs信號傳導(dǎo)途徑上游基因ghbri1表達水平略有上升,ghbki1表達水平無明顯變化;該途徑下游基因ghbsu1和ghbin2表達水平上調(diào),而ghbzr1表達水平下降。下調(diào)ghmakr3表達,ghbri1表達水平下降,ghbki1表達水平上升;而ghbsu1和ghbin2表達水平下降,ghbzr1表達水平上升。這表明ghmakr3基因表達影響brs信號傳導(dǎo)途徑相關(guān)基因的表達,可能參與brs信號傳導(dǎo)過程。4.ghmakr3基因?qū)w維發(fā)育相關(guān)的myb轉(zhuǎn)錄因子表達的影響qrt-pcr分別檢測超量和抑制ghmakr3表達的轉(zhuǎn)基因棉花中與纖維發(fā)育相關(guān)的myb轉(zhuǎn)錄因子的表達變化。上調(diào)ghmakr3表達后ghmyb25-like、ghmyb25、ghmyb109等基因表達水平降低,且開花當天胚珠表皮纖維細胞起始密度低于同時期的野生型。下調(diào)ghmakr3表達后除ghmyb109表達水平上升外,ghmyb25-like、ghmyb25表達水平無明顯變化,同時開花當天胚珠表皮纖維細胞起始密度無明顯變化。5.下調(diào)ghmakr3基因可促進糖向種子的轉(zhuǎn)運上調(diào)ghmakr3基因表達后,成熟種子可溶性總糖含量變化不明顯,而蔗糖轉(zhuǎn)運蛋白基因ghsut1-9表達水平不同程度降低;下調(diào)ghmakr3基因表達后成熟種子可溶性總糖含量較野生型提高,同時ghsut1-9表達水平有不同程度的上升。表明抑制ghmakr3的表達可以促進糖向種子的轉(zhuǎn)運。6.下調(diào)ghmakr3基因促進了棉鈴及種子的發(fā)育觀察10dpa棉鈴發(fā)現(xiàn),超量表達ghmakr3基因后棉鈴與苞葉均比同時期野生型變小,而干擾該基因表達后棉鈴與苞葉均變大。考種和纖維檢測分析得出,上調(diào)GhMAKR3基因表達棉花結(jié)鈴性降低、棉鈴和種子變小;而下調(diào)GhMAKR3基因表達能夠促進棉鈴的生長發(fā)育,使結(jié)鈴數(shù)增多、棉鈴和種子變大。上述結(jié)果表明,GhMAKR3基因具有棉鈴優(yōu)勢表達特性,上調(diào)其表達抑制棉鈴的生長發(fā)育;反之,干擾該基因的表達則促進棉鈴的生長發(fā)育。推測GhMAKR3的表達可能與BRs水平存在負相關(guān)性,即上調(diào)該基因表達與降低BRs水平的表型類似,而抑制其表達與提高BRs水平的表型類似。
[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.
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S562

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1 劉通;棉鈴優(yōu)勢表達基因GhMAKR3對棉鈴發(fā)育的影響[D];西南大學(xué);2016年

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本文編號：2150647