摘要
棉花是一种重要的经济作物，在国民经济中具有重要作用。当前我国棉花生产中存在两个关键问题：一是棉花黄萎病危害严重，二是棉纤维品质需进一步改良。针对以上问题，本论文从基因的角度进行了以下三部分研究：
(1) 海岛棉(Gossypium barbadense)防卫反应相关基因GbRar1、GbSgt1，GbRac1的克隆及转基因烟草的抗病性分析。
Rar1、Sgt1、Rac1是三个R防卫反应途径的基因，功能定位于R蛋白下游与活性氧激发之间。用简并引物PCR及3’RACE等技术，从海岛棉品种7124中克隆了GbRar1、GbSgt1、GbRac1等三种基因。Southern杂交证明GbRar1、GbRac1在海岛棉基因组中都以单拷贝形式存在。Northern blot表明，三种基因经棉花黄萎病菌诱导后，mRNA表达量显著增加，暗示GbRar1、GbSgt1、GbRac1可能参与海岛棉对黄萎病菌的防卫反应。
构建了植物表达载体pSgt、pRar和pRac，用根瘤农杆菌LBA4404分别转化烟草品种NC89，Southern杂交证明GbRar1、GbSgt1、GbRac1基因都已分别整合到烟草基因组中。采用离体叶片接种法，用烟草赤星病菌对转基因植株进行抗病性鉴定，以非转基因烟草为对照，结果显示转GbSgt1、GbRar1、GbRac1基因的烟草分别有5株、7株、4株抗性明显提高。
据我们所知，目前尚没有Rar1和Sgt1在转基因植物中抗病性分析的报道，本实验为植物防卫反应相关蛋白在植物抗病基因工程中的应用进一步提供了实验证据。
(2) 海岛棉GbKTN1基因的克隆和过量表达对酵母细胞伸长的影响。
KTN1是一种微管切断蛋白，已知拟南芥KTN1在纤维细胞起始伸长、持续延伸及细胞壁合成过程中起重要作用。用5’RACE/3’RACE从海岛棉7124开花后10d的纤维细胞中分离获得了GbKTN1的cDNA序列。Southern杂交证明GbKTN1在海岛棉基因组中以单拷贝形式存在。半定量RT-PCR结合Southern杂交分析表明，GbKTN1在海岛棉根、茎、叶及纤维细胞中均有表达，以纤维细胞中的表达最高，叶中最低。推测GbKTN1在厚壁组织细胞中的表达要高于其它类型的细胞，但这尚需用实验进一步证明。
利用裂殖酵母系统，初步分析了GbKTN1对细胞伸长的功能，发现该基因在酵母细胞中过量表达使细胞长度增加2～3倍，部分细胞呈不规则形状，这暗示GbKTN1在海岛棉中可能具有调控纤维细胞伸长的功能。对GbKTN1与纤维细胞
①8
细胞壁合成和细胞伸长的关系作进一步的深入研究，揭示它与棉纤维发育的相关
性，可能会为棉花纤维品质改良的基因工程提供一些有益的思路。
门）中棉（Gos地ium arboreum）光诱导基因 Gacab启动子的克隆及其功
能研究。
高等植物的捕光叶绿素a儿蛋白复合体基因（ca）是一类光诱导表达的基
因。利用加接头法，从金华中棉中分离获得了Gacab编码区5’上游的调控序列
1009 hp，命名为 Gacab P，与公布的其它植物 cab启动子序列比较，没有明显
的同源性。将 Gacab P和 197、504、779 hp的 5’端缺失体分别与 gus（md A）
基因融合，构建植物表达载体。GUS组织化学分析 Gacab P：：gus在烟草不同
器官中的稳定表达，发现 Gacab P驱动 gus基因在叶片及绿色幼嫩组织中表达。
在暗培养的转基因烟草叶片中 Gacab P不表现活性，而光能够诱导 gus基因的
表达。这些结果表明，Gacab P是一种光诱导型启动子。用基因枪轰击水稻愈伤
组织，结果显示 Gacab P及三种不同长度的缺失体均可驱动 gus基因的瞬时表
达，以 504 hp缺失体活性最高，197 hp、779 hp和 1009 hp的表达减弱。由此
推测 Gacab基因的基本启动子存在于－197一fop，－504一197 hp间包含
Gacab启动子的正调控元件，－1009一504 hp间包含负调控元件。另外，504 hp
缺失体驱动 gSS的瞬时表达水平明显高于 3 5 S启动子，这一现象值得进一步探
讨。根据目前文献报道，本文获得的序列是从棉属植物中分离得到的第一个 cab 5’
上游调控序列，这为植物基因工程与光调控的研究提供了可用的元件。
Cotton, as one of the most important economic crops, has played an important role in our national economy development. Currently two key problems are facing in cotton production in China that is the prevention from infection of Verticllium wilt and the upgrade of fiber quality. Targeting to solve these two problems at a gene level, three parts of studies have been carried out in this thesis as following:
(1) Cloning of GbRar1, GbSgt1, and GbRacl genes from Gossypium barbadense and analysis of disease resistance of transgenic tobacco plants
The coding sequence of the three defense signaling components, Rar1, Sgt1 and Racl proteins, known act at a position between downstream of R gene and oxidative burst, was cloned respectively from G. barbadense. Southern blot analysis indicated GbRarl and GbRacl genes were existed in G barbadense as a single copy. Northern-blot showed the level of mRNAs of GbRarl, GbSgtl or GbRac1 was dramatically increased upon induction with Verticllium dehliae inoculation. It suggests that the GbRar1, GbSgt1 or GbRacl may involve in the defense response of G. barbadense to Verticllium wilt infection.
Three plant expression vectors pSgt, pRar and pRac, harboring GbRar1, GbSgtl or GbRacl gene driven by CaMV 35S promoter, were constructed and used for transformation of Nicotiana tabacum var. NC89, respectively. Southern-blot demonstrated that all these three genes were integrated into the genome of tobacco. Disease challenge test of leaves of transgenic plants in vitro by inoculation of Alternaria alternata showed that the resistance was enhanced compared with the non-transgenic plants. This implicates that they may have a potential application in genetic engineering of plants with enhanced disease resistance.
(2) Cloning of GbKTN1 gene from G. barbadense and effect of GbKTNl over-expression on yeast cell elongation
Katanin is a protein with a function of coupled ATP hydrolysis and microtubule severing. It is well known that the homologous gene coding for katanin-like protein in Arabidopsis has a significant role in cell elongation and cell wall biosynthesis. The cDNA of GbKTNl gene was isolated from 10 DPA fiber cells of G. barbadense using 5'RACE/3'RACE techniques. Southern-blot hybridization indicates it is a single copy
gene in G.barbadense. Semi-quantitative RT-PCR combined with southern-blot analysis revealed that GbKTN1 was expressed in roots, hypocotyls, leaves and fibers. However, the transcripts mRNA of GbKTNl was most abundant in fiber cells, while it was lowest in leaves. It is likelihood that GbKTN1 gene is preferentially expressed in the sclerenchyma cells than that in other types of cells.
The GbKTNl cDNA was transformed into S. pombe to confirm its function on cell elongation. Results showed that most yeast cells over-expressing GbKTNl gene were elongated dramatically with a cell length of 2-3 fold increase than that of non-transformed cells. Its implication is the GbKTN1 gene may correlate with the cell elongation in G.barbadense.
(3) Cloning and functional analysis of a light-inducible Gacab promoter from Gossypium arboreum
A 1009 bp promoter sequence of cab gene encodes chlorophyll a/b binding protein belonging to a class of light-inducible proteins was cloned from G. arboreum. Sequence analysis showed no obvious homology to the published cab promoters. The full-length Gacab P and 5' end deletions with a length of 197 bp, 504 bp or 779 bp was fused with gus (uid A) gene respectively and plant expression vectors constructed. Construct with Gacab P:: gus was used for transformation of Nicotiana tabacum var. NC89 and transgenic tobacco plants generated. GUS histochemical assay showed that GUS was specifically expressed in leaves and young green tissues. GUS was not detected in leaves of transgenic plants grown in the dark for 6d, whereas GUS was highly expressed in leaves of these plants further induced with light for 6d, demonstrating Gacab P was a light-inducible promoter. Transit GUS expression in rice calli indicated that the expression level of 504 bp
引文
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