棉花膜联蛋白基因在逆境胁迫或纤维发育中的功能解析

发布时间：2018-05-24 23:10

  本文选题：棉花 + 膜联蛋白　； 参考：《南京农业大学》2016年博士论文

【摘要】：棉花是世界性重要的经济作物,棉纤维是世界上最重要的天然纺织原料。尽管棉花是一种相对耐逆的作物,但是随着气候变化及环境恶化,高盐和干旱等逆境胁迫严重制约着棉花产量与品质,同时人民生活水平提高和植棉机械化的需求,对优质纤维品质指标的要求也越来越高。如何在国家18亿亩耕地的红线下,确保棉花产量及品质的提高成为当前棉花生产面临的严峻挑战。进一步探究棉花纤维发育分子机理,利用现代分子生物学技术及遗传工程发掘并探究耐逆、优质纤维品质相关基因作用机理,培育抗逆、优质、高产棉花新品种是应对这一挑战的最高效、经济的途径。本实验室前期通过同源克隆的方法,从陆地棉纤维cDNA文库中分别获得了两个典型的膜联蛋白基因,分别编码316和314个氨基酸,命名为GhAnn1(Genbank No:KM062523 )和GhFAnnxA(Genbank No: ACJ11719)。本研究在此基础上对两个膜联蛋白基因进行更深入的结构、功能、机理解析。组织器官表达模式分析发现GhAnn1为组成型表达基因,在营养器官根、茎、叶及胚珠和纤维发育不同阶段表达量均处于较高水平,而GhFAnnxA为纤维特异表达基因,主要在棉花开花后5天到20天的纤维(5dpa-20dpa)中表达。进一步与拟南芥、水稻等物种中的膜联蛋白进化分析发现,GhAnn1与拟南芥膜联蛋白基因AtAnn1表现出较高的同源性,而GhFAnnxA则与AnxGb6显示较高的同源性。前人报道AtAnn1在干旱和盐胁迫中起重要作用,AnxGb6在棉花纤维伸长中起重要作用。因此,我们分别对GhAnn1基因在逆境中的功能及GhFAnnxA基因在棉花纤维发育中的功能进行了系统解析。一、GhAnn1在逆境(干旱和高盐)中的功能解析体内和体外亚细胞定位结果均表明,GhAnn1蛋白位于细胞膜。GhAnn1作为一个膜定位的蛋白,为感受并响应逆境胁迫提供了重要的基础。进一步激素、逆境(干旱和高盐)及H202诱导表达分析发现,受ABA、JA、H2O2、高盐及干旱等诱导后,GhAnn1表达量显著增加,而SA处理4、6、10h后,其表达量也显著提高,表明GhAnn1在棉花逆境胁迫中扮演者重要角色。通过农杆菌介导的棉花遗传转化获得转基因阳性植株,经多代自交、纯合,分别获得6个过表达、6个反义及6个3'端特异反义转基因纯系。通过对GhAnn1转基因纯系萌发期和苗期的表型鉴定发现,虽然在200mM盐胁迫和PEG6000 ( 15%)模拟干旱胁迫条件下,野生型与转基因株系萌发、生长等均受到抑制,但是与野生型相比,萌发期GhAnn1转基因过表达株系萌发率显著提高、根系显著变长;苗期GhAnn1转基因过表达株系长势更加旺盛,根系生长更加茂密,株高更高,而转基因抑制表达纯系无论在萌发期还是苗期均表现出相反的表型。在干旱胁迫处理后20天,与野生型相比,GhAnn1转基因过表达株系气孔完整性更好、气孔开度变大、失水率变小,而GhAnn1转基因抑制表达纯系则相反,表明GhAnn1在棉花抗旱耐盐中起重要作用。基于气孔开度的变化,利用1μMABA对野生型及GhAnn1棉花转基因植株叶片进行处理,通过气孔开度调查发现,GhAnn1转基因过表达株系对ABA变的更加敏感,推测GhAnn1响应逆境可能与ABA途径调控的逆境信号通路有关。为了进一步确定GhAnn1基因对抗旱耐盐的作用,对200mM盐胁迫和PEG6000(15%)模拟干旱胁迫条件下野生型及GhAnn1转基因纯系植株叶片中的生理指标进行了检测。结果显示:与野生型相比,GhAnn1转基因过表达株系叶绿素、游离脯氨酸和可溶性糖含量显著增加、SOD酶活显著提高、MDA含量显著降低,而在GhAnn1转基因抑制表达纯系中则呈现相反的结果,表明GhAnn1通过提高棉花植株的光合、渗透及抗氧化胁迫能力,进而提高棉花抗旱耐盐性。二、GhFAnnxA在棉花纤维发育中的功能解析根据GhFAnnxA在基因组的位置,对GhFAnnxA物理距离5Mb以内的已报道棉花纤维品质QTL进行整合分析,发现在此区段内含有棉花纤维长度、强度、马克隆值等性状相关的QTL。对3年(2007-2009年)3个不同地区(安阳、库车与南京)277个不同来源棉花品种的自然群体进行基因与性状关联分析,发现GhFAnnxA与纤维长度、强度显著相关,表明GhFAnnxA在纤维发育中起重要作用。通过对GhFAnnxA转基因纯系纤维表型分析发现,与野生型相比,GhFAnnxA过表达株系成熟纤维长度变长,而干扰株系则显著变短。扫描电镜(SEM)分析发现纤维变长是由于棉花纤维发育伸长引起的,与起始期无关。进化及表达分析发现GhFAnnxA影响纤维发育可能与Ca2+和H202密切相关。为了进一步探究GhFAnnxA如何影响纤维伸长,首先通过胚珠离体培养技术发现Ca2+在纤维发育中的作用并且外源Ca2+比内源Ca2+在纤维发育中起更重要的作用。通过对GhFAAnxA转基因纯系进行Fluo-3/AM组织化学染色发现,与野生型相比,GhFAnnxA过表达纯系OE-2和OE-5纤维中Ca2+含量增高,而RNAi干扰纯系RNAi-2和RNAi-3中Ca2+含量明显降低,表明GhFAnnxA影响纤维发现是通过影响Ca2+含量实现的。对RNAi干扰材料进行体外恢复实验表明GhFAnnxA可能作为Ca2+通道影响Ca2+的内流,进而影响纤维发育。进一步分析发现Ca2+并不直接影响纤维发育,而是通过影响ROS来实现的。首先通过胚珠离体培养技术证明H2O2在纤维发育中起重要作用,而高浓度的H2O2抑制纤维伸长,通过对纤维中ROS组织化学染色及H2O2含量的测定,发现ROS的产生依赖于Ca2+。进一步对GhFAnnxA转基因材料中ROS组织化学染色及H2O2含量测定分析发现,与野生型相比,GhFAnnxA过表达纯系OE-2和OE-5纤维中ROS及H2O2含量增高,而RNAi干扰纯系RNAi-2和RNAi-3中含量明显降低,表明GhFAnnxA影响了 ROS的产生进而影响了纤维发育。通过对GhFAnnxA转基因过表达纯系体外抑制试验及RNAi干扰材料的恢复试验对这一结论进行了进一步验证。考虑到ROS在纤维发育中的重要性,进一步探究ROS来源研究。研究发现,第一,当加入不同浓度的DPI (NADPH酶抑制剂)时,纤维细胞中ROS和H2O2的产量明显受到抑制;第二,不同浓度DPI条件下胚珠离体培养纤维细胞中NADPH酶活受到明显的抑制;第三,与野生型相比,GhFAnnxA转基因过表达株系OE-2和OE-5中NADPH酶活显著上升,而RNAi干扰株系RNAi-2和RNAi-3中则显著下降。上述结果表明ROS的产生可能来源于NADPH氧化酶(Rboh)。通过对陆地棉TM-1进行全基因组扫描,结合进化及蛋白结构分析我们筛选出2个Rboh候选基因,分析发现这两个基因受Ca2+及DPI诱导表达,其编码的蛋白位于细胞膜;与野生型相比,GhFAnnxA转基因过表达株系OE-2和OE-5中,这2个基因在纤维发育不同时期表达量显著上升,而RNAi干扰株系RNAi-2和RNAi-3中则显著下降。表明这2个Rboh基因对转基因材料中ROS产生起重要作用并且与Ca2+相关。过量的ROS能够抑制纤维发育,那么如何保证纤维发育过程中ROS的平衡呢?分析发现GhCDPK1可以在调控水平上与GhPOX1和GhAPX1共同维持纤维细胞内ROS的平衡。首先,通过对GhFAnnxA转基因材料伸长期纤维中GhPOX1和GhAPX1表达变化分析发现GhPOX1和GhAPX1在ROS的平衡中起重要作用。其次,我们通过全基因扫描获得陆地棉中全部CDPKs,进而通过进化及表达分析选取GhD10G2029(GhCDPK1)作为候选基因,分析发现GhCDPK1受Ca2+诱导表达,在纤维发育中的表达模式与GhFAnnxA存在一致性;与野生型相比,GhFAnnxA转基因过表达株系OE-2和OE-5中GhCDPK1在纤维发育不同时期表达量显著上升,而在RNAi干扰株系RNAi-2和RNAi-3中则显著下降,表明GhCDPAK1在调节ROS平衡中可能起重要作用。通过Y2H和BiFC互作分析发现GhCDPK1可以与Rboh全长和N端进行互作,并且随着Ca2+浓度的提高,互作强度越弱,表明GhCDPK1可以对Rbohs进行调控,但是这种调控受Ca2+的影响。通过对纤维伸长阶段相关基因(蔗糖合酶基因、K+转运蛋白、糖转运蛋白及Expansin基因)及次生壁加厚时期相关基因(纤维素合酶、几丁质酶及1,3-β-葡聚糖酶)在野生型及GhFAnnxA转基因材料纤维中的表达分析,发现GhFAnnxA通过影响细胞内膨压和细胞壁松弛相关基因表达,进而影响纤维伸长和次生壁加厚。除此之外,研究发现与野生型相比,虽然Actin微纤丝的形态没有发生显著变化,GhFAnnxA过表达纯系OE-2和OE-5中,Actin微纤丝在棉花纤维细胞中的占有率均显著增长,而干扰表达纯系RNAi-2和RNAi-3 Actin微纤丝在棉花纤维细胞中的占有率则显著降低,表明GhFAnnxA影响Actin微纤丝的含量,进而影响纤维发育。Y2H和BiFC互作分析发现,GhFAnnxA可以与Actin互作,同时GhFAnnxA与Actin共定位于整个内膜系统和细胞核,这一结果为GhFAnnxA与Actin互作提供了基础。通过对GhFAnnxA转基因纯系和野生型棉花纤维进行LatB (Actin去聚合化试剂)处理分析发现,与野生型相比,GhFAnnxA过表达纯系OE-2和OE-5中,纤维细胞Actin微丝聚合能力显著增强,而干扰表达纯系RNAi-2和RNAi-3, Actin微丝聚合能力显著下降。同时,在GhFAnnxA过表达及干扰表达纯系中,驱动蛋白GhKCH1和GhKCH2表达变化与Actin微丝聚合能力变化表现一致。结果表明GhFAnnxA影响纤维伸长不仅与Actin的聚合和动态变化有关,还与微管具有密切的关系。
[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.
【学位授予单位】：南京农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S562
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本文编号：1930985