發(fā)布時間：2018-05-24 23:10

  本文選題：棉花 + 膜聯(lián)蛋白��； 參考：《南京農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：棉花是世界性重要的經(jīng)濟(jì)作物,棉纖維是世界上最重要的天然紡織原料。盡管棉花是一種相對耐逆的作物,但是隨著氣候變化及環(huán)境惡化,高鹽和干旱等逆境脅迫嚴(yán)重制約著棉花產(chǎn)量與品質(zhì),同時人民生活水平提高和植棉機(jī)械化的需求,對優(yōu)質(zhì)纖維品質(zhì)指標(biāo)的要求也越來越高。如何在國家18億畝耕地的紅線下,確保棉花產(chǎn)量及品質(zhì)的提高成為當(dāng)前棉花生產(chǎn)面臨的嚴(yán)峻挑戰(zhàn)。進(jìn)一步探究棉花纖維發(fā)育分子機(jī)理,利用現(xiàn)代分子生物學(xué)技術(shù)及遺傳工程發(fā)掘并探究耐逆、優(yōu)質(zhì)纖維品質(zhì)相關(guān)基因作用機(jī)理,培育抗逆、優(yōu)質(zhì)、高產(chǎn)棉花新品種是應(yīng)對這一挑戰(zhàn)的最高效、經(jīng)濟(jì)的途徑。本實驗室前期通過同源克隆的方法,從陸地棉纖維cDNA文庫中分別獲得了兩個典型的膜聯(lián)蛋白基因,分別編碼316和314個氨基酸,命名為GhAnn1(Genbank No:KM062523 )和GhFAnnxA(Genbank No: ACJ11719)。本研究在此基礎(chǔ)上對兩個膜聯(lián)蛋白基因進(jìn)行更深入的結(jié)構(gòu)、功能、機(jī)理解析。組織器官表達(dá)模式分析發(fā)現(xiàn)GhAnn1為組成型表達(dá)基因,在營養(yǎng)器官根、莖、葉及胚珠和纖維發(fā)育不同階段表達(dá)量均處于較高水平,而GhFAnnxA為纖維特異表達(dá)基因,主要在棉花開花后5天到20天的纖維(5dpa-20dpa)中表達(dá)。進(jìn)一步與擬南芥、水稻等物種中的膜聯(lián)蛋白進(jìn)化分析發(fā)現(xiàn),GhAnn1與擬南芥膜聯(lián)蛋白基因AtAnn1表現(xiàn)出較高的同源性,而GhFAnnxA則與AnxGb6顯示較高的同源性。前人報道AtAnn1在干旱和鹽脅迫中起重要作用,AnxGb6在棉花纖維伸長中起重要作用。因此,我們分別對GhAnn1基因在逆境中的功能及GhFAnnxA基因在棉花纖維發(fā)育中的功能進(jìn)行了系統(tǒng)解析。一、GhAnn1在逆境(干旱和高鹽)中的功能解析體內(nèi)和體外亞細(xì)胞定位結(jié)果均表明,GhAnn1蛋白位于細(xì)胞膜。GhAnn1作為一個膜定位的蛋白,為感受并響應(yīng)逆境脅迫提供了重要的基礎(chǔ)。進(jìn)一步激素、逆境(干旱和高鹽)及H202誘導(dǎo)表達(dá)分析發(fā)現(xiàn),受ABA、JA、H2O2、高鹽及干旱等誘導(dǎo)后,GhAnn1表達(dá)量顯著增加,而SA處理4、6、10h后,其表達(dá)量也顯著提高,表明GhAnn1在棉花逆境脅迫中扮演者重要角色。通過農(nóng)桿菌介導(dǎo)的棉花遺傳轉(zhuǎn)化獲得轉(zhuǎn)基因陽性植株,經(jīng)多代自交、純合,分別獲得6個過表達(dá)、6個反義及6個3'端特異反義轉(zhuǎn)基因純系。通過對GhAnn1轉(zhuǎn)基因純系萌發(fā)期和苗期的表型鑒定發(fā)現(xiàn),雖然在200mM鹽脅迫和PEG6000 ( 15%)模擬干旱脅迫條件下,野生型與轉(zhuǎn)基因株系萌發(fā)、生長等均受到抑制,但是與野生型相比,萌發(fā)期GhAnn1轉(zhuǎn)基因過表達(dá)株系萌發(fā)率顯著提高、根系顯著變長;苗期GhAnn1轉(zhuǎn)基因過表達(dá)株系長勢更加旺盛,根系生長更加茂密,株高更高,而轉(zhuǎn)基因抑制表達(dá)純系無論在萌發(fā)期還是苗期均表現(xiàn)出相反的表型。在干旱脅迫處理后20天,與野生型相比,GhAnn1轉(zhuǎn)基因過表達(dá)株系氣孔完整性更好、氣孔開度變大、失水率變小,而GhAnn1轉(zhuǎn)基因抑制表達(dá)純系則相反,表明GhAnn1在棉花抗旱耐鹽中起重要作用。基于氣孔開度的變化,利用1μMABA對野生型及GhAnn1棉花轉(zhuǎn)基因植株葉片進(jìn)行處理,通過氣孔開度調(diào)查發(fā)現(xiàn),GhAnn1轉(zhuǎn)基因過表達(dá)株系對ABA變的更加敏感,推測GhAnn1響應(yīng)逆境可能與ABA途徑調(diào)控的逆境信號通路有關(guān)。為了進(jìn)一步確定GhAnn1基因?qū)�抗旱耐鹽的作用,對200mM鹽脅迫和PEG6000(15%)模擬干旱脅迫條件下野生型及GhAnn1轉(zhuǎn)基因純系植株葉片中的生理指標(biāo)進(jìn)行了檢測。結(jié)果顯示:與野生型相比,GhAnn1轉(zhuǎn)基因過表達(dá)株系葉綠素、游離脯氨酸和可溶性糖含量顯著增加、SOD酶活顯著提高、MDA含量顯著降低,而在GhAnn1轉(zhuǎn)基因抑制表達(dá)純系中則呈現(xiàn)相反的結(jié)果,表明GhAnn1通過提高棉花植株的光合、滲透及抗氧化脅迫能力,進(jìn)而提高棉花抗旱耐鹽性。二、GhFAnnxA在棉花纖維發(fā)育中的功能解析根據(jù)GhFAnnxA在基因組的位置,對GhFAnnxA物理距離5Mb以內(nèi)的已報道棉花纖維品質(zhì)QTL進(jìn)行整合分析,發(fā)現(xiàn)在此區(qū)段內(nèi)含有棉花纖維長度、強(qiáng)度、馬克隆值等性狀相關(guān)的QTL。對3年(2007-2009年)3個不同地區(qū)(安陽、庫車與南京)277個不同來源棉花品種的自然群體進(jìn)行基因與性狀關(guān)聯(lián)分析,發(fā)現(xiàn)GhFAnnxA與纖維長度、強(qiáng)度顯著相關(guān),表明GhFAnnxA在纖維發(fā)育中起重要作用。通過對GhFAnnxA轉(zhuǎn)基因純系纖維表型分析發(fā)現(xiàn),與野生型相比,GhFAnnxA過表達(dá)株系成熟纖維長度變長,而干擾株系則顯著變短。掃描電鏡(SEM)分析發(fā)現(xiàn)纖維變長是由于棉花纖維發(fā)育伸長引起的,與起始期無關(guān)。進(jìn)化及表達(dá)分析發(fā)現(xiàn)GhFAnnxA影響纖維發(fā)育可能與Ca2+和H202密切相關(guān)。為了進(jìn)一步探究GhFAnnxA如何影響纖維伸長,首先通過胚珠離體培養(yǎng)技術(shù)發(fā)現(xiàn)Ca2+在纖維發(fā)育中的作用并且外源Ca2+比內(nèi)源Ca2+在纖維發(fā)育中起更重要的作用。通過對GhFAAnxA轉(zhuǎn)基因純系進(jìn)行Fluo-3/AM組織化學(xué)染色發(fā)現(xiàn),與野生型相比,GhFAnnxA過表達(dá)純系OE-2和OE-5纖維中Ca2+含量增高,而RNAi干擾純系RNAi-2和RNAi-3中Ca2+含量明顯降低,表明GhFAnnxA影響纖維發(fā)現(xiàn)是通過影響Ca2+含量實現(xiàn)的。對RNAi干擾材料進(jìn)行體外恢復(fù)實驗表明GhFAnnxA可能作為Ca2+通道影響Ca2+的內(nèi)流,進(jìn)而影響纖維發(fā)育。進(jìn)一步分析發(fā)現(xiàn)Ca2+并不直接影響纖維發(fā)育,而是通過影響ROS來實現(xiàn)的。首先通過胚珠離體培養(yǎng)技術(shù)證明H2O2在纖維發(fā)育中起重要作用,而高濃度的H2O2抑制纖維伸長,通過對纖維中ROS組織化學(xué)染色及H2O2含量的測定,發(fā)現(xiàn)ROS的產(chǎn)生依賴于Ca2+。進(jìn)一步對GhFAnnxA轉(zhuǎn)基因材料中ROS組織化學(xué)染色及H2O2含量測定分析發(fā)現(xiàn),與野生型相比,GhFAnnxA過表達(dá)純系OE-2和OE-5纖維中ROS及H2O2含量增高,而RNAi干擾純系RNAi-2和RNAi-3中含量明顯降低,表明GhFAnnxA影響了 ROS的產(chǎn)生進(jìn)而影響了纖維發(fā)育。通過對GhFAnnxA轉(zhuǎn)基因過表達(dá)純系體外抑制試驗及RNAi干擾材料的恢復(fù)試驗對這一結(jié)論進(jìn)行了進(jìn)一步驗證�？紤]到ROS在纖維發(fā)育中的重要性,進(jìn)一步探究ROS來源研究。研究發(fā)現(xiàn),第一,當(dāng)加入不同濃度的DPI (NADPH酶抑制劑)時,纖維細(xì)胞中ROS和H2O2的產(chǎn)量明顯受到抑制;第二,不同濃度DPI條件下胚珠離體培養(yǎng)纖維細(xì)胞中NADPH酶活受到明顯的抑制;第三,與野生型相比,GhFAnnxA轉(zhuǎn)基因過表達(dá)株系OE-2和OE-5中NADPH酶活顯著上升,而RNAi干擾株系RNAi-2和RNAi-3中則顯著下降。上述結(jié)果表明ROS的產(chǎn)生可能來源于NADPH氧化酶(Rboh)。通過對陸地棉TM-1進(jìn)行全基因組掃描,結(jié)合進(jìn)化及蛋白結(jié)構(gòu)分析我們篩選出2個Rboh候選基因,分析發(fā)現(xiàn)這兩個基因受Ca2+及DPI誘導(dǎo)表達(dá),其編碼的蛋白位于細(xì)胞膜;與野生型相比,GhFAnnxA轉(zhuǎn)基因過表達(dá)株系OE-2和OE-5中,這2個基因在纖維發(fā)育不同時期表達(dá)量顯著上升,而RNAi干擾株系RNAi-2和RNAi-3中則顯著下降。表明這2個Rboh基因?qū)�轉(zhuǎn)基因材料中ROS產(chǎn)生起重要作用并且與Ca2+相關(guān)。過量的ROS能夠抑制纖維發(fā)育,那么如何保證纖維發(fā)育過程中ROS的平衡呢?分析發(fā)現(xiàn)GhCDPK1可以在調(diào)控水平上與GhPOX1和GhAPX1共同維持纖維細(xì)胞內(nèi)ROS的平衡。首先,通過對GhFAnnxA轉(zhuǎn)基因材料伸長期纖維中GhPOX1和GhAPX1表達(dá)變化分析發(fā)現(xiàn)GhPOX1和GhAPX1在ROS的平衡中起重要作用。其次,我們通過全基因掃描獲得陸地棉中全部CDPKs,進(jìn)而通過進(jìn)化及表達(dá)分析選取GhD10G2029(GhCDPK1)作為候選基因,分析發(fā)現(xiàn)GhCDPK1受Ca2+誘導(dǎo)表達(dá),在纖維發(fā)育中的表達(dá)模式與GhFAnnxA存在一致性;與野生型相比,GhFAnnxA轉(zhuǎn)基因過表達(dá)株系OE-2和OE-5中GhCDPK1在纖維發(fā)育不同時期表達(dá)量顯著上升,而在RNAi干擾株系RNAi-2和RNAi-3中則顯著下降,表明GhCDPAK1在調(diào)節(jié)ROS平衡中可能起重要作用。通過Y2H和BiFC互作分析發(fā)現(xiàn)GhCDPK1可以與Rboh全長和N端進(jìn)行互作,并且隨著Ca2+濃度的提高,互作強(qiáng)度越弱,表明GhCDPK1可以對Rbohs進(jìn)行調(diào)控,但是這種調(diào)控受Ca2+的影響。通過對纖維伸長階段相關(guān)基因(蔗糖合酶基因、K+轉(zhuǎn)運蛋白、糖轉(zhuǎn)運蛋白及Expansin基因)及次生壁加厚時期相關(guān)基因(纖維素合酶、幾丁質(zhì)酶及1,3-β-葡聚糖酶)在野生型及GhFAnnxA轉(zhuǎn)基因材料纖維中的表達(dá)分析,發(fā)現(xiàn)GhFAnnxA通過影響細(xì)胞內(nèi)膨壓和細(xì)胞壁松弛相關(guān)基因表達(dá),進(jìn)而影響纖維伸長和次生壁加厚。除此之外,研究發(fā)現(xiàn)與野生型相比,雖然Actin微纖絲的形態(tài)沒有發(fā)生顯著變化,GhFAnnxA過表達(dá)純系OE-2和OE-5中,Actin微纖絲在棉花纖維細(xì)胞中的占有率均顯著增長,而干擾表達(dá)純系RNAi-2和RNAi-3 Actin微纖絲在棉花纖維細(xì)胞中的占有率則顯著降低,表明GhFAnnxA影響Actin微纖絲的含量,進(jìn)而影響纖維發(fā)育。Y2H和BiFC互作分析發(fā)現(xiàn),GhFAnnxA可以與Actin互作,同時GhFAnnxA與Actin共定位于整個內(nèi)膜系統(tǒng)和細(xì)胞核,這一結(jié)果為GhFAnnxA與Actin互作提供了基礎(chǔ)。通過對GhFAnnxA轉(zhuǎn)基因純系和野生型棉花纖維進(jìn)行LatB (Actin去聚合化試劑)處理分析發(fā)現(xiàn),與野生型相比,GhFAnnxA過表達(dá)純系OE-2和OE-5中,纖維細(xì)胞Actin微絲聚合能力顯著增強(qiáng),而干擾表達(dá)純系RNAi-2和RNAi-3, Actin微絲聚合能力顯著下降。同時,在GhFAnnxA過表達(dá)及干擾表達(dá)純系中,驅(qū)動蛋白GhKCH1和GhKCH2表達(dá)變化與Actin微絲聚合能力變化表現(xiàn)一致。結(jié)果表明GhFAnnxA影響纖維伸長不僅與Actin的聚合和動態(tài)變化有關(guān),還與微管具有密切的關(guān)系。
[Abstract]:Cotton is the most important economic crop in the world. Cotton fiber is the most important natural textile raw material in the world. Although cotton is a relatively resistant crop, with the climate change and environmental deterioration, high salt and drought stress seriously restrict the yield and quality of cotton, the improvement of people's living standard and the demand for mechanization of cotton planting in the same time. The demand for quality fiber quality indicators is becoming higher and higher. How to ensure cotton yield and quality under the red line of 1 billion 800 million acres of cultivated land is a severe challenge for cotton production. Further explore the molecular mechanism of cotton fiber development, explore and explore the resistance and quality with modern molecular biology technology and genetic engineering. The mechanism of fiber quality related genes and the cultivation of new varieties of resistant, high quality and high yield cotton are the most efficient and economical way to deal with this challenge. In the early stage of the laboratory, two typical egg white egg white genes were obtained from the land cotton fiber cDNA library by homologous cloning method, which encode 316 and 314 amino acids respectively, named Gh. Ann1 (Genbank No:KM062523) and GhFAnnxA (Genbank No: ACJ11719). Based on this study, the two annexin genes were further structured, functional, and mechanism parsing. Tissue organ expression pattern analysis found that GhAnn1 was a component expression gene and expressed in vegetative organs root, stem, leaf and ovule and fiber development at different stages. At a higher level, GhFAnnxA is a fibrous specific expression gene expressed mainly in the fiber (5dpa-20dpa) 5 days to 20 days after the flowering of cotton. Further analysis with the evolution of annexin in Arabidopsis, rice and other species found that GhAnn1 and Arabidopsis membrane associated protein gene AtAnn1 showed high homology, while GhFAnnxA was shown to be with AnxGb6. High homology. Predecessors reported that AtAnn1 played an important role in drought and salt stress and AnxGb6 played an important role in cotton fiber elongation. Therefore, we systematically analyzed the function of GhAnn1 gene in adversity and the function of GhFAnnxA gene in the development of cotton fiber. 1, the function of GhAnn1 in adversity (drought and high salt). The results of subcellular localization in vivo and in vitro showed that GhAnn1 protein located in the cell membrane.GhAnn1 as a membrane protein, provided an important basis for sensing and responding to stress stress. Further hormones, adversity (drought and high salt) and H202 induced expression analysis found that GhAnn1 expression was induced by ABA, JA, H2O2, high salt and drought. The amount of SA was significantly increased, and the expression of 4,6,10h increased significantly, indicating that GhAnn1 played an important role in cotton stress stress. Transgenic positive plants were obtained through genetic transformation of Agrobacterium tumefaciens, and 6 overexpressions, 6 antisense and 6 3'terminal specific antisense transgenic lines were obtained by Agrobacterium mediated genetic transformation. The phenotypic identification of the GhAnn1 transgenic pure line at the germination stage and the seedling stage showed that, although under 200mM salt stress and PEG6000 (15%) simulated drought stress, the growth of the wild type and the transgenic lines were inhibited, but compared with the wild type, the germination rate of the GhAnn1 transgenic overexpressed plant lines increased significantly and the root system grew significantly longer than the wild type. The growth of GhAnn1 transgenic plants was more vigorous, the root growth was thicker, and the plant height was higher, while the transgenic inhibition expression lines showed the opposite phenotype in both germination and seedling stage. Compared with the wild type 20 days after drought stress treatment, the stomatal integrity and stomatal opening of GhAnn1 transgenic lines were better. The water loss rate was smaller, while the GhAnn1 transgenic inhibition expression was the opposite, indicating that GhAnn1 played an important role in the drought resistance and salt tolerance of cotton. Based on the change of stomatal opening, the leaves of transgenic plants of wild type and GhAnn1 cotton were treated by 1 micron MABA. Through the stomatal opening investigation, it was found that the GhAnn1 transgenic overexpressed strain was more sensitive to ABA change. In order to further determine the effect of the GhAnn1 gene on the drought and salt tolerance, the physiological indexes in the leaves of the wild type and GhAnn1 transgenic pure line plants under the condition of 200mM salt stress and PEG6000 (15%) simulated drought stress were detected in order to further determine the effect of the GhAnn1 gene on the control of the drought and salt tolerance. The results showed that the physiological indexes of the wild type and GhAnn1 transgenic plant leaves under the condition of simulated drought stress were detected by 200mM salt stress and PEG6000 (15%). In contrast, the content of chlorophyll, free proline and soluble sugar in GhAnn1 transgenic lines increased significantly, the activity of SOD enzyme increased significantly, and the content of MDA decreased significantly, while in the pure lines of GhAnn1 transgenic inhibition, the results showed that GhAnn1 increased the photosynthesis, osmotic and antioxidant stress of cotton plant plants, and then increased the ability to increase the resistance to oxidative stress. Resistance to drought and salt tolerance of cotton. Two, GhFAnnxA in cotton fiber development function analysis based on the location of GhFAnnxA in the genome, the integrated analysis of reported cotton fiber quality QTL within the GhFAnnxA physical distance 5Mb, found that the length, strength, horse clone value of cotton fiber length, strength, and the value of horse cloned value in this area are 3 years (2007-2009). In 3 different regions (Anyang, Kuche and Nanjing), 277 natural populations of cotton varieties from different sources were related to the genetic correlation analysis. It was found that GhFAnnxA was significantly related to fiber length and strength, indicating that GhFAnnxA played an important role in the development of fiber. By phenotypic analysis of GhFAnnxA transgene pure fiber, it was found that compared with the wild type, GhF The length of mature fiber in AnnxA overexpressed line was longer, but the interference strain was significantly shorter. The scanning electron microscope (SEM) analysis found that fiber length was caused by the growth elongation of cotton fiber, which was not related to the beginning period. Evolution and expression analysis found that GhFAnnxA affects the development of fiber may be closely related to Ca2+ and H202. In order to further explore GhFAnnxA such as What affects fiber elongation, first through the ovule vitro culture technique to discover the role of Ca2+ in the development of fiber and the more important role of exogenous Ca2+ than endogenous Ca2+ in the development of fiber. By Fluo-3/AM histochemical staining of GhFAAnxA transgenic lines, it is found that GhFAnnxA over expressed C in pure line OE-2 and OE-5 fiber. The content of a2+ increased, while the content of Ca2+ in the RNAi interference pure RNAi-2 and RNAi-3 decreased obviously. It showed that GhFAnnxA influenced the fiber discovery by affecting the Ca2+ content. In vitro recovery experiment on the RNAi interference material showed that GhFAnnxA may affect the Ca2+ internal flow as Ca2+ channel, and then affect the development of fiber. Further analysis found Ca2+ is not. The development of the fiber is directly affected by the influence of ROS. First, through the ovule isolation, it is proved that H2O2 plays an important role in the development of fiber, and the high concentration of H2O2 inhibits the elongation of fiber. By the determination of the ROS histochemical staining and the content of H2O2 in the fiber, it is found that the production of ROS is dependent on Ca2+. to further the GhFAnnxA transformation. ROS histochemical staining and H2O2 content determination analysis showed that the content of ROS and H2O2 in GhFAnnxA overexpressed OE-2 and OE-5 fibers was higher than that of wild type, while RNAi interference in pure RNAi-2 and RNAi-3 content decreased obviously, indicating that GhFAnnxA affects the production of ROS and then affects the development of fiber. This conclusion was further verified by the expression of pure line in vitro inhibition test and RNAi interference material recovery. Considering the importance of ROS in the development of fiber, the study of ROS sources was further explored. First, when different concentrations of DPI (NADPH inhibitor) were added, the production of ROS and H2O2 in the fiber cells was obviously suppressed. Second, under the condition of different concentrations of DPI, the NADPH activity of the ovule cultured in vitro was significantly inhibited, and third, compared with the wild type, the NADPH activity of the GhFAnnxA transgenic overexpressed line OE-2 and OE-5 increased significantly, while the RNAi interfering strain was significantly decreased in RNAi-2 and RNAi-3. The results indicated that the ROS production may come from NADP. H oxidase (Rboh). We screened 2 Rboh candidate genes by whole genome scanning of upland cotton TM-1, combined with evolution and protein structure analysis. It was found that the two genes were induced by Ca2+ and DPI, and their encoded proteins were located in the cell membrane. Compared with the wild type, the GhFAnnxA transgenic overexpressed strains were 2 genes in OE-2 and OE-5. In different periods of fiber development, the amount of expression increased significantly, while the RNAi interference strain RNAi-2 and RNAi-3 decreased significantly. It indicates that these 2 Rboh genes play an important role in the production of ROS in the transgenic materials and are related to Ca2+. The excess ROS can inhibit the development of the fibers. How to ensure the balance of ROS in the process of fiber development? Analysis found GhCDP K1 can maintain the balance of ROS in fiber cells together with GhPOX1 and GhAPX1 at the regulatory level. First, it is found that GhPOX1 and GhAPX1 play an important role in the balance of ROS through the analysis of the GhPOX1 and GhAPX1 expression changes in the elongated fibers of GhFAnnxA transgenic materials. Secondly, we obtain all CDPKs in upland cotton by full gene scanning. GhD10G2029 (GhCDPK1) was selected as candidate gene through evolution and expression analysis. It was found that GhCDPK1 was induced by Ca2+, and the expression pattern in fiber development was consistent with GhFAnnxA. Compared with wild type, the expression of GhCDPK1 in GhFAnnxA transgenic lines OE-2 and OE-5 increased significantly at different periods of fiber development in OE-2 and OE-5, but in RN. The Ai interference strain decreased significantly in RNAi-2 and RNAi-3, indicating that GhCDPAK1 may play an important role in regulating ROS balance. Through the interaction of Y2H and BiFC, it is found that GhCDPK1 can interact with Rboh and N ends, and with the increase of Ca2+ concentration, the weaker the interaction strength, indicating that GhCDPK1 can regulate it, but this regulation is regulated. The expression of the related genes (sucrose synthase gene, K+ transporter, sugar transporter and Expansin gene) and the secondary wall thickening period related genes (cellulose synthase, chitinase and 1,3- beta glucan enzyme) in the wild type and GhFAnnxA transgenic material fibers were analyzed by 2+. It was found that GhFAnnxA was affected by the influence of thin film on the fiber of the wild and GhFAnnxA transgenic materials. Intracellular expansion and cell wall relaxation related gene expression, and then influence fiber elongation and secondary wall thickening. In addition, the study found that compared with the wild type, although the morphology of Actin microfibrils did not change significantly, GhFAnnxA overexpressed pure line OE-2 and OE-5, Actin microfibril had a significant increase in the cotton fiber cell occupancy. The interference expression of pure line RNAi-2 and RNAi-3 Actin microfibrils in cotton fiber cells decreased significantly, indicating that GhFAnnxA affects the content of Actin microfibrils, and then affects the interaction between.Y2H and BiFC in fiber development. It is found that GhFAnnxA can interact with Actin, while GhFAnnxA and Actin are located in the whole intima system and the nucleus. The results provided a basis for the interaction of GhFAnnxA and Actin. Through the analysis of LatB (Actin depolymerization reagent) treatment of GhFAnnxA transgenic and wild cotton fibers, it was found that GhFAnnxA overexpressed pure line OE-2 and OE-5, and the Actin microfilament polymerization ability of fibrous cells increased significantly compared with the wild type, and the interference expressed pure line RNAi-2 and RNAi-3. The polymerization ability of Actin microfilament decreased significantly. At the same time, the expression of GhKCH1 and GhKCH2 in the GhFAnnxA overexpressed and interfered pure lines was consistent with the change of Actin microfilament polymerization ability. The results showed that the effect of GhFAnnxA on the elongation of fiber was not only related to the polymerization and dynamic modification of Actin, but also closely related to microtubule.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S562
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本文編號：1930985