【作者】 韩杰；

【导师】 张献龙；

【作者基本信息】 华中农业大学 ， 作物遗传育种， 2014， 博士

【摘要】 棉花是重要的经济作物,棉花纤维是最重要的天然纺织工业原料。棉花纤维是由棉花胚珠外表皮的单细胞发育而成的表皮毛,从纤维细胞的突起到成熟的纤维需要45到60天,纤维的长度主要由快速伸长期决定。植物肽类激素PSK是一种能够从植物细胞内分泌到胞外磺化五肽,能由细胞膜上特异的受体感知,进而引起一系列生理生化反应,PSK信号在植物细胞伸长过程中起重要作用。PSK的作用机理研究较多,但在应用于作物对产量或品质进行改良的研究至今还未见报道。PSK能促进细胞伸长,其是否能促进棉花纤维伸长而改良棉花纤维品质值得探究。本研究首先通过胚珠培养体系证明了外源添加PSK-α能够显著促进棉花纤维的伸长,进而克隆了棉花PSK基因,通过转基因得到了纤维品质在长度和细度方面得到改良的超表达转基因棉花,并对PSK信号在棉花纤维发育调控机理方面进行了系统的分析研究。具体研究结果如下：1、在胚珠培养体系中外源添加PSK能够促进棉花纤维的伸长。在胚珠培养体系中,低浓度的PSK-α就能够显著的促进棉花纤维的伸长,这种促进作用存在着剂量效应,1μmol·L-1是最优浓度。在含有GA3和IAA培养基中外源添加PSK-α (1μmol·L-1)对受精后的胚珠进行培养,纤维长度能够增加27%,在不含IAA的培养基中添加PSK-α,其对纤维伸长的促进效果更加显著,纤维长度能够增加46%。PSK-α对未受精胚珠纤维发育的促进更加显著。氯酸盐(KClO3)是多肽磺化的抑制剂,添加到胚珠培养的培养基中能够抑制内源PSK-α的产生而抑制胚珠和纤维发育。外源添加PSK-α能够部分的恢复KClO3对纤维发育的抑制作用,进一步证实PSK的确能促进纤维发育。2、棉花PSK前体基因GhPSK的克隆和序列分析。通过同源序列法从陆地棉纤维中克隆了一个棉花PSK基因,该基因编码一个85氨基酸的PSK前体多肽,其氨基酸序列的C端包含了成熟PSK五肽序列(YIYTQ)。 GhPSK和拟南芥的PSK4、PSK5相似度较高,和玉米、水稻的PSK序列差异较大。GhPSK在开花后5到20天的纤维细胞中优势表达,在下胚轴、根和花瓣中也有着高量表达。为了验证GhPSK的功能,在拟南芥中异源表达GhPSK,转基因拟南芥主根长度显著增加,而且增加的程度和ChPSK的表达水平正相关。3、在棉花中超表达GhPSK毙够改良纤维的长度和细度。将GhPSK构建RNAi干涉载体和35S启动子驱动的超表达载体,通过农杆菌介导法获得转基因棉花。GhPSK在超表达株系中的表达丰度显著提高,但是在RNAi株系中干涉程度并不明显。考察田间和温室T2代株系纤维长度,GhPSK超表达较野生型显著增长,但RNAi朱系纤维长度变化不明显。追踪纤维发育进程发现超表达GhPSK能够在快速伸长期显著增加纤维伸长速率。进一步考察T3代GhPSK超表达纯系纤维性状发现超表达GhPSK不仅能够提高纤维长度,还显著改良纤维的细度。4、GhPSK通过调节K+稳态和ROS水平促进纤维伸长。通过液质联用技术对10DPA纤维中内源PSK-α进行定量,发现超表达GhPSK能够显著提高纤维内源PSK-α的水平。根据已有文献报道,PSK信号促进纤维的伸长可能是通过调节胞内K+的水平,因此利用非损伤微测技术对GhPSK超表达株系和野生型对照10DPA纤维尖端的K+流进行检测,结果表明超表达株系纤维尖端的K+外流速率显著低于野生型。为了验证GhPSK是否与K+有关,通过胚珠培养体系来检测超表达GhPSK对外源K+反应,在低浓度和适宜浓度的K+处理下,GhPSK超表达株系的纤维长度较野生型显著增加,然而在高浓度的K+条件下,二者纤维长度的差异不明显。这些结果说明了PSK-α信号可以通过直接或间接的方式影响纤维细胞中K+的稳态,进而影响纤维的伸长。进一步采用Solexa测序来研究GhPSK调控纤维发育的路径,对比超表达株系和野生型(10DPA纤维)表达谱,发现有132个差异表达基因。PSK信号还可能通过影响调节细胞ROS水平的基因表达从而促进纤维细胞的快速伸长。5、PSK受体基因GhPSKR的克隆、序列分析以及转基因后代分析。以拟南芥的PSKR序列进行BLAST,在棉花EST数据库搜索到一条同源序列ChPSKR,通过5’-RACE扩增到一条3296bp具有完整ORF的cDNA,其编码1013个氨基酸,具有膜定位信号肽、富亮氨酸结构域(LRR)、PSK结合结构域、跨膜结构域和受体激酶结构域等典型的PSK受体结构域。该基因和蓖麻、拟南芥和胡萝卜的PSKR相似性最高,而和水稻中的PSKI有一定的差异。GhPSKR在棉花的根、下胚轴和次生壁合成期的纤维细胞内表达丰度较高,在叶片、胚珠和伸长期纤维中也有着一定量的表达。将GhPSKR构建RNAi干涉载体、35S启动子和GbEXPA2启动子分别驱动的超表达载体,通过农杆菌介导得到了转基因棉花,通过Southern blot和qRT-TCR筛选出了单拷贝表达量变化较为显著的株系,为下一步系统的研究PSK信号在纤维发育中调控机理提供了材料。更多还原

【Abstract】 Cotton is an important economic crop that produces the most important natural fiber in the textile industry. Cotton fibers are single-celled trichomes derived from the ovule epidermis. The fiber development lasts45-60days from initials to mature fibers, and the rapid elongation stage determines the fiber length. Phytosulfokine-a (PSK-a) is a disulfated autocrine pentapeptide, and is perceived by special plasma membrane-localized LRR receptors, which will induce some series of physiological responses in plants. In recent years, numerous studies have focused on the mechanism of PSK, however, the application of PSK in crop genetic improvement has not been reported. PSK signaling plays an important role in plant cell elongation and cotton fibers are extremely elongated cells, so it is likely that PSK could improve cotton fiber yield and quality. To investigate the role of PSK signaling involved in cotton fiber development, we first demonstrated that exogenous PSK-a could promote cotton fiber elongation in ovule culture system, and cloned cotton PSK gene GhPSK, then obtained GhPSK overexpression transgenic cotton lines with longer and finer fibers. Furthermore, the regulation mechanism of PSK signalling in cotton fiber development was also investigated. The main results are as following:1. Exogenous PSK-a promotes fiber elongation in vitro.In the cotton ovule culture system, very low levels of PSK-a were found to promote cotton fiber production, but the effect was dose-dependent, a concentration of1μmol·L-1was the optimal concentration. The fertilized ovules (0DPA) treated with PSK-a (1μmol·L-1) enhanced fiber length by27%in the present of GA3+IAA. Additionally, in the medium lacking IAA (GA3+PSK-α), fiber length increased by46%compared to the control (GA3alone), which suggested that exogenous PSK-a promotes fiber elongation more significantly in the absence of IAA. To unfertilized ovules (-1DPA), this improvement was more significant. Potassium chlorate (KClO3), as an effective inhibitor of peptide sulfation, could suppress endogenous PSK-a production in the ovule and fiber cells. Addition of1μmol·L-1PSK-α in the ovule culture medium only partly rescued the KC103-dependent inhibition of fiber development, which suggested that the PSK really improve the fiber development. 2. Molecular cloning and characterization of the GhPSK gene.By homologous sequences method, a putative full-length cotton PSK gene was cloned from upland cotton, using developing fiber cDNA as the template. This gene encoded a polypeptide of85amino acids that is predicted to be a phytosulfokine (PSK) precursor, which contained a highly conserved single PSK domain (YIYTQ) close to the C terminus. Sequence alignment revealed that GhPSK was more similar to PSK4and PSK5of Arabidopsis than the ones from maize and rice in the evolutionary relationships. GhPSK is preferentially expressed in5to20DPA cotton fibers and in hypocotyl, root and petal. To validate the function of GhPSK, transgenic Arabidopsis lines were obtained, the root lengths of GhPSK overexpression lines showed significant increases, and the increases were positively correlated with the expression levels.3. Overexpression of GhPSK improves cotton fiber length and fineness.RNAi silencing construct pHG4-PSK and overexpression construct CaMV35S::PSK were obtained and introduced into cotton line YZ1via Agrobacterium-mediated transformation. The expression levels of GhPSK was highly enhanced in overexpression lines but was slightly decreased in RNAi lines. Mature fibers of overexpression lines in field and greenhouse exhibited a significant increase in the length in T2generation, but there were no significant changes for RNAi lines. Additionally, to track when the observed changes occur, the fiber lengths in different development stages were investigated, fiber lengths of GhPSK overexpression lines were significantly increased compared to wild type during the fiber rapid elongation stage. The fiber quality of the GhPSK overexpression homozygous lines in T3generation was further evaluated in field trials. The results showed that mature fiber length and fineness significantly improved in the overexpression lines.4. PSK signaling promotes cotton fiber elongation through the regulation of K+and ROShomeostasis.Endogenous PSK-a concentration of10DPA fibers was measured by LC/MS. As expected, due to overexpression of the GhPSK, the endogenous PSK-a levels in10DPA fibers of overexpression lines increased significantly, comparing to wild type. According to the reports, PSK signaling may facilitate fiber elongation by regulating intracellular K+levels, so K+flux at the tips of10DPA cotton fiber cells was measured by the non-invasive scanning ion-selective electrode technique. The mean K+efflux rates in the overexpression lines were significantly lower than wild type. Furthermore an experiment was designed to examine the relationship between fiber elongation and K+, which was to investigate the fiber developments with additions of different concentrations of K+into the ovule culture medium. After7days culture, the fiber lengths in GhPSK-overexpressing plants were increased significantly compared to wild type at low and intermediate concentrations of K+, however, the enhancement rate of fiber length was significantly reduced at the high K+concentration. These results lead us to believe that PSK-a exerts an effect on K+homeostasis, in a direct or indirect manner. Digital Gene Expression (DGE) profiles analysis was used to identify genes that were differentially expressed in10DPA fibers between GhPSK overexpression lines and wild-type. In total132differentially expressed genes were identified, and PSK signaling might also regulate fiber cell rapid elongation by affecting the expression of the genes involved in the regulation of ROS level.5. Molecular cloning, characterization and genetic transformation of the GhPSKRA putative GhPSKR EST had been found from cotton EST database with Arabidopsis PSKR as the reference sequence. The full-length cDNA sequence of3296bp was cloned by5’-RACE, and it encoded1013amino acids, which contained NH2-terminal hydrophobic signal sequence, extracellular leucine-rich repeats (LRRs), PSK binding domain, trans-membrane domain and cytoplasmic kinase domain. GhPSKR was more similar to PSKR from Ricinus communis, Daucus Carota and Arabidopsis, but different from Rice. GhPSKR was preferentially expressed in15-20DPA cotton fibers, hypocotyls and roots. It also expressed in leaves, ovules and elongating fibers. The RNAi construct and overexpression constructs driven by35S and GbEXPA2promoters were introduced into upland cotton line YZ1via Agrobacterium-mediated transformation. Several positive transgenic lines with single copy insert and enhanced GhPSKR expression levels were identified by Southern blotting and qRT-PCR, which were the materials to elucidate PSK signalling pathway in cotton fiber development.更多还原