【作者】 秦超；

【导师】 范玲； 曲延英；

【作者基本信息】 新疆农业大学 ， 作物遗传育种， 2009， 硕士

【摘要】 棉花是世界上最重要的天然纤维作物,棉花在我国国民经济中占有十分重要的地位。近年来,克隆与棉纤维次生壁加厚发育相关的基因,从分子水平改良棉纤维品质已成为主要研究方向。肉桂酰辅酶A还原酶(Cinnamoyl-CoA Reductase,CCR)是苯丙烷代谢途径中的一个关键酶,此酶可能对苯丙烷代谢途径的碳流具有潜在的调控作用。为了明确棉花CCR基因的基本功能和对木质素含量的影响,我们利用已经从棉花中克隆的木质素生物合成的限速酶基因—肉桂酰辅酶A还原酶基因(GhCCR4),构建其瞬时表达载体,通过基因枪法转化棉花胚珠,研究基因表达对棉纤维发育的影响。然后利用农杆菌浸染法转化棉花,分析了GhCCR4的功能。实验结果如下:1)实验构建了由CaMV35S启动子驱动的pGUS-CCR4融合表达载体,使用基因枪轰击法将其转化棉花胚珠,确定了转化0 DPA胚珠的最佳条件:轰击压力为1350 psi,轰击距离为9 cm,轰击次数为2次。GUS组织化学染色结果表明,GhCCR4基因在棉花纤维伸长期和次生壁增厚期持续表达。不同发育时期纤维长度测量结果发现,在8 d时转GhCCR4基因纤维长度和对照相比没有明显差别,但在27 d时纤维长度明显短于对照。纤维透射电镜切片观察发现,转GhCCR4基因的细胞次生壁与对照相比增厚达17 %。上述结果说明,GhCCR4基因在纤维细胞中的表达对棉花纤维的伸长起到了抑制作用并能够使细胞壁次生壁在一定程度上增厚。2)对棉花茎尖农杆菌介导转化的多个因素进行了比较,完善了的农杆菌茎尖转化体系。在以农杆菌介导的棉花转化过程中,应以2天的无菌苗茎尖作为农杆菌的感染受体,激素的最佳配比浓度为KT 0.5 mg/L、IAA 0.1 mg/L,茎尖长势相对较好,出芽率最高;卡那霉素的最适筛选浓度为50 mg/L;扦插生根的方法能够缩短再生植株生育周期。使用上述方法,筛选抗性转化率达到35%以上,转化周期缩短至2-3个月。3)利用农杆菌介导法获得了批量的抗性幼苗,为避免假阳性植株的出现,首先进行了卡那霉素点涂和GUS化学检测,获得阳性植株;以CaMV35S启动子和NPTⅡ报告基因序列设计引物,进行阳性植株PCR扩增,PCR检测的阳性率在30%左右。使用抗性筛选和分子检测相结合的方法,获得了12株转基因棉花。同时利用半定量PCR方法进行了转录水平的检测,发现他们与对照之间存在较为明显的差异,反义抑制的效率较高,抑制了CCR基因的表达。4)改进了提取棉花不同时期纤维的RNA的热硼酸法。棉花GhCCR4基因在本文所检测的组织中均有所表达,尤其在根,茎杆中表达量较大,说明此基因有组织表达特异性。在棉纤维不同发育时期的转录水平有很大变化,在纤维发育初期(0-10 DPA)表达微弱,在15,20 DPA表达量最大,在25 DPA表达量降低,说明此基因在棉纤维伸长发育后期高效表达。5)对GhCCR4基因过量表达和RNA干扰转基因棉花苗期形态观察、木质素和纤维素含量检测等方法进行比较和分析,发现他们与对照之间在植株形态上存在一定的差异,木质素含量降低,这可能的原因是外源基因抑制了基因的表达。本研究为从分子水平改良纤维品质提供候选目的基因,为研究棉花纤维发育调控的分子机理提供理论基础。更多还原

【Abstract】 Cotton is the world’s leading fiber crop and is a mainstay of economy in China, which is the largest cotton producer and consumer around the world. Fiber is the major product of cotton production and the main raw material in textile industry. Cinnamoyl-CoA reductase (CCR, EC 1.2.1.44), one of the key enzymes in the first step of the phenylpropanoid pathway, catalyzes the NADPH-dependent reduction of cinnamoyl-CoA esters to their corresponding cinnamaldehydes. A gene encoding Gossypium hirsutum Cinnamoyl-CoA reductase 4 (GhCCR4) was isolated from cotton fiber. Here we examine the role of GhCCR4 in cotton development.1) We choose transient expression assay system for test of GhCCR4 function in cotton fibers by using cotton ovule culture and biolistic transformation techniques. The transient expression vector pGUS-CCR4 is driven by CaMV35S promoter with GUS reporter gene and GhCCR4 gene. The conditions for bombardment were optimized : the highest transformation efficiency of GUS was obtained when 0 day of anthesis (0DPA) ovules were applied, bombardment include helium pressures of 1350 psi, distance of 9 cm and bomb times of twice. Histochemical staining showed that high levels of GhCCR4 gene expression was detected during the fiber rapid elongation stage and the secondary wall thickening stage in cultured ovules. Measurement of fiber length in different developmentally stage showed that the fiber length of the transgenic plants at the stage (8 DPA) was not different as compared to that of the wild-type plants . The fiber length in the transgenic plants reduced 19% compared with wild-type plants at the stage of 27 DPA. The cell wall of the transgenic fibre at the stage of 27 DPA was thicked as compared to that of the wild-type fibre. Transmission electron microscopy demonstrated that the wall thickness of transgenic fibre was increased to 17% of that of the wild type. These findings suggest that GhCCR4 could play a critical role in the processes of elongation and secondary cell wall formation during fibre development.2) An improved transformation system of meristem via Agrobacterium-mediated transformation was established after studies on many influential factors. The transformation rate of anti-antibiotic reached 35% and the transformation cycle decreased to 2-3 months. Incorporated anti-antibiotic and molecular detection, Selective Strategy was upgraded after improving selection of transformation plants. Transgenic plants were obtained and some of them had been carried out their target-gene measurements in leaves. All the results showed that the state of the tip conditions and the Selective Strategy acted vital roles in transformation rate and cycle.3) GhCCR4 were mediated into the cotton genome in identical direction via Agrobacterium-mediated transformation. After pre-culture、co-culture、selective culture、sub-culture and root induction culture, neogenesis young shoots with kanamycin resistance were obtained finally. The PCR primers were designed to depend on the gene order of CaMV35S and NPTⅡ. Results of PCR indicated that the target gene had integrated into the genome successfully. The detection of transcription level indicated that they have visible different between transgenetic plant and control. The efficiency of sense enhancement and RNA interference is high in them, and exogenous gene restrain the expression of CCR.4) The hot borate method for efficient isolation of cotton total RNA were improved in different cotton fibers. Cotton total RNA was isolated from different tissues of immature plants and expression of these genes was analyzed by RT-PCR. The expression of GhCCR4 gene was detected of every tissue in this article. GhCCR4 gene is expressed high in root and stipe. This experiments showed a differential expression pattern of mRNA levels in different cotton fiber. Expression of gene in other tissues and each tissue from different growth period will be further studied.5) Transgenetic palnts of both sense enhancement and RNA interference were compared and analyzed through appearance viewing, lignin and cellulose content mensuration. The result show that it have some difference between them in morphous. The content of lignin cut down. The possible reason is the exogenous gene repressed the CCR expression.Based on this data, we suggest that GhCCR4 may play an important role in the morphogenesis and secondary wall thickening of cotton by positively/negatively regulating the structure of cell wall in growing course. Our study presents important experimental evidence for the function of lignin and provides gene candidates for genetic improvement of cotton fiber quality.更多还原