【作者】 谢永芳；

【导师】 王伯初；

【作者基本信息】 重庆大学 ， 生物医学工程， 2008， 博士

【摘要】 植物次生代谢物质与其贮藏器官-腺体(或腺毛)关系密切相关。了解次生代谢物质贮藏器官发生与形成的基因网络及其调控机制,能为实践中有效的调控植物次生代谢提供理论依据。其中,棉属(gossypium)植物的棉酚与其贮藏器官腺体(gland)是研究次生代谢工程较理想的一个研究模式。棉酚(Gossypol)为锦葵科棉族(Gossypieae)植物特有,存在于色素腺体(pigment gland)中,是棉花自身重要的抗性物质,但由于棉酚对人类和单胃动物有毒,限制了丰富的棉籽蛋白及油脂资源的利用。为有效调控棉酚合成,极有必要对色素腺体形成的分子机制进行研究。因此本文从以下几个方面进行相关研究:首先选用川2802(种子、植株都有腺体),湘棉18(种子无腺体,植株有腺体,种子萌发过程中腺体逐渐增多。),无腺体棉-显无N5(种子、植株都无腺体)为材料,对腺体形成时期不同品种的棉花的腺体特点进行观察,并检测次生代谢产物棉酚的含量变化及相关抗氧化酶活性的变化规律。其次,利用三种不同材料的腺体差别,通过棉花基因芯片,对腺体形成时期的基因进行高通量筛选,筛选表达变化的基因。最后以“湘棉18”特殊腺体为材料,构建了其腺体形成时期的均一化全长cDNA文库。根据芯片结果,在所建cDNA文库中进行腺体形成时期相关基因的克隆,获得腺体形成时期相关候选基因后,结合生物信息学方法对所克隆的基因功能进行预测,并对其在不同部位的时空表达进行了研究,从而为揭示棉花腺体形成的分子机制提供了重要的理论依据。实验研究结果如下:1.棉花的腺体形成及相关生理指标研究湘l8种子在萌发48h前后,色素腺体开始形成,川2802在种子萌发36h前后,新的色素腺体逐渐形成。腺体形成前后直径逐渐由小变大,其后色素腺体数目有较大幅度的增长,色素腺体直径变化不大。川棉2802在种子萌发初期其棉酚含量逐渐降低,萌发5d后其棉酚含量缓慢升高;而湘棉18(色素延缓形成棉子)在种子萌发时期,其棉酚含量呈缓慢增长趋势,并逐渐接近有色素棉川棉2802的棉酚含量。在棉花种子在萌发初期,可溶性糖含量呈总体下降趋势,萌发一周后,可溶性糖含量逐渐呈上升趋势。可溶性蛋白含量在早期含量较高,随之逐渐含量减低,萌发一周后,含量逐渐升高。在棉花种子萌发进程中,过氧化物酶、过氧化氢酶、超氧化物歧化酶的活性呈上升趋势,其中川棉2802的活性相对最高,湘棉18次之,显无N5的活性较低。2.棉花腺体发育过程中的基因表达变化的高通量分析本研究利用高通量的方法分析了棉花腺体形成过程中的基因表达变化。采用包含大约23,977个基因探针的Affymetrix棉花基因芯片,以湘棉18突变体、野生棉(川2802)、无腺体棉(显无N5)为研究材料,对腺体形成过程中差异表达的基因进行了鉴定。结果显示,在腺体形成过程中,川棉(2802)、湘棉18腺体形成后、湘棉18腺体形成前三种材料中的共同有差异表达的基因311条,其中112条上调,191条下调;湘棉18腺体形成后、川棉(2802)、无腺体棉N5的共同差异表达的基因有549条,其中218条上调,331条下调。上述基因主要涉及蛋白质聚合、微管运动、脂类合成、氨基酸磷酸化、代谢途径调控的酶基因、受外界胁迫表达的抗逆相关基因、光合作用相关基因、转录调控因子等以及其它一些未知功能的基因。采用RT-PCR验证了其中6个基因,均与芯片结果一致。从中可以推测,腺体的形成是多因素多基因共同作用的结果,它们通过相互调节并形成调控网络,从而直接或间接地导致腺体的形成。3.湘棉18腺体形成时期均一化全长cDNA文库构建:构建了湘棉18腺体形成时期的均一化全长cDNA文库,主要采用SMART (Switching mechanism at 5 end of RNA transcript)技术与DSN技术(Duplex-specific nuclease )相结合的方法,即mRNA逆转录后合成cDNA,经均一化处理,Sfi I酶切,连接在质粒载体,电转化,最后获得5.86×105个独立克隆,重组率高达94% ,插入片段的平均长度约为1.4 kb,从而成功建立棉花腺体形成相关基因的cDNA文库。通过均一化文库的建立,使文库中高丰度基因拷贝数有了很大程度的降低,而低丰度基因的丰度得到相对提升,从而大大增加了从文库中克隆发现新基因的概率,为克隆腺体形成相关基因奠定基础。4.棉花腺体形成相关的候选基因GAP1的克隆和分析从基因芯片筛选的与腺体形成相关的基因中,选取DW513956设计引物,对均一化全长文库进行PCR筛选,获得全长序列,命名为GAP1(GeneBank收录号:EU3703012)。GAP1基因全长为1203 bp的cDNA序列,开放阅读框起始位点为25bp处,终止位点为771bp处,编码248个氨基酸。蛋白分子量为33.09 KD,理论等电点pI为8.99,亚细胞定位在胞质内,GAP1属于疏水分泌性蛋白质,有一个信号肽,具分泌型过氧化物酶的保守区域。与烟草的过氧化物酶,红豆的过氧化物酶,大戟等的相似度较高,分别为75%,75%,73%。对GAP1基因,进一步应用原位杂交的方法分析其表达的具体部位,发现其mRNA的表达主要集中在植株的纤维化程度高的部位,如导管、厚壁细胞等位置,在薄壁细胞处表达量少。5.腺体形成相关的候选基因GhZIF的克隆和分析从基因芯片筛选的与腺体形成相关的基因中,选取DT463838进行了深入分析。首先对均一化全长文库进行PCR筛选,获得全长序列,命名GhZIF(GeneBank收录号:EU3703033)。GhZIF基因全长为1989 bp的cDNA序列,起始位点为135bp处,终止位点为1598bp处,编码487个氨基酸。蛋白分子量为53.75KD,理论等电点pI为5.79,亚细胞定位在质膜上,为疏水性,跨膜锚定蛋白,具转运蛋白保守区域。对GhZIF基因,其mRNA的时空表达研究发现,在腺体形成前有表达,在腺体形成后表达信号弱,在成熟植株细胞中,其mRNA的表达广泛,在薄壁细胞与维管束细胞都有分布。通过本研究,为了解腺体形成的分子机制奠定了坚实的基础,为从分子水平上认识棉花腺体和棉酚的相互关系开辟新的途径,具有理论意义和实践应用价值。更多还原

【Abstract】 The content of secondary metabolite is closely related to the quantity of storage organ. Study on the molecular mechanism under the development of the storage organ can provide the theoretical direction for the effective agricultural practice. As the result, it is meaningful to study the correlation between the secondary metabolites and storage organ. As is well known, Gossypol and gland of cotton have been a good model for the research of genetic engineering.Gossypol, a secondary metabolite stored in the glands of cotton, protecting cottonseed from consumption of human and monogastric animal. More interestingly, this ability is unique to the tribe Gossypieae. Although the relationship between gossypol and pigment gland has been studied for a long time, the development mechanism of pigment gland has not been investigated at molecular level.In this study, the material we used is Xiangmian18, a mutant which had the special property of delayed pigment gland morphogenesis, i.e., the glands in the leave appeared to be normal in the plant, but the seeds have low gossypol level. Firstly,we studyed on the characteristics of gland development and detected the changes of the gossypol content and antioxidant enzyme activity. Secondly, differentially expressed genes were screened by high throughput screening technology based on difference in gland of cotton. Furthermore,we established a normalized and full-length cDNA library of its pigment formation phase and cloned candidate genes related gland formation followed by bioinformatics analysis of the selected genes. The results may pave the way for the further investigation the molecular mechanism of gland formation and the identification of genes directly related to gland formation.The main results of this research are as follows:1.The observation to gland formation and study on the resistance-related physiological parameters in cotton.The glands were formated after 48h germination in xiangmian18. In chuanmian2802 new gland grew after 36h germination. The diameter became bigger during the gland foramtion.The content of gossypol decreased during the early germination and rised slowly after 5d gernmination in chuanmian2802. The content showed a slow body growth tend in xiangmian18, consistent with chuanmian2802.The content of soluble suger and the content of soluble proteins tended to decrease in general during early germination of cotton and rise gradually after a weekly germination.The activity of POD,CAT and SOD increased during germination of cotton. The activities of antioxidant enzyme were highest in chuanmian2802, xiangmian18 was in second place, and xianwuN5 showed low activites.2. High through-out analysis of pigment gland formation related genesTo reveal features of cotton gland development, the transcriptomes on several mutantants during the gland development have been compared. We studied gene expression profiles of Xiangmian18, chuanmian2802, XianwuN5 during cotton gland development using Affymetrix cotton GeneChip array that contains probes for about 23,977 genes. Comparative analysis among the post-gland formation, the undevelopment gland of Xiangmian 18 and chuan2802, the samples showed that 112 genes were up and 191 were down-regulated. Comparative analysis among chuanmian 2802, the post-gland formation, Xiangwu N5, the samples showed that 218 genes were up and 331 were down-regulated. These genes were classified into six major GO categories relevant to such biological processes as protein polymerization, microtubule-based, lipid biosynthetic process movement, response to oxidative stress, photosynthesis gene. Among them, 6 genes have been further confirmed with RT-PCR and the results were in accordance with the Genechip’s data. These genes were regulated by each other, playing roles directly or indirectly in the gland formation of cotton.3. Establishment of a normalized and full-length cDNA library at the stage of pigment gland formationA normalized cDNA library was constructed from upland cotton during its pigments gland forming age with SMART (Switching mechanism at 5 end of RNA transcript) technique and DSN technique (Duplex-specific nuclease). mRNAs were isolated from upland cotton;Then double-stranded cDNAs were synthesized from mRNAs and processed by normalization; digested with SfiI; and then the cDNAs were ligated to pDNR-LIB vector. The ligation mixture was transformed into E.coli JM109 by electroporation. Tittering results revealed that approximately 5.86×105 independent clones were included in this library. Electrophoresis gel results fragments ranged from 800 bp to 2 kb, with an average size of 1400 bp. Random picking clones showed that the recombination rate was 94%. Our successfully constructed cDNA library is a full-length library with high efficiency, and can be used to identify the forming genes related to development of pigments gland cottons.After normalizing, abundant classes of different genes tended to equalize, which can increase the chance to discover new genes.4 Cloning and analysis on the representative pigment development related gene GAP1.Based on the result of screening cotton microarray, the primers were designed according to DW513956. Using PCR to screen the constructed library, we got full length cDNA, named GAP1 (gland related protein1, GenBank number: EU3703012) The complete nucleotide sequence of GAP1 cDNA has 1203 bp and contains a long open reading frame of 747 bp. The predicted protein sequence derived from the open reading frame produces a 248-amino acid polypeptide, with a calculated molecular mass of 33.09 KD and a theoretical iso-electric point of 8.99; Protein subcellular location was predicted in the cytoplasm; the protein is hydrophobic property with a signal peptide, a peroxidase domain. There were 75%, 75%,73% homologic comparison with Nicotiana tabacum, Vigna angularis, Euphorbia pekinensis respectively.In situ hybridization was performed to analyze the expression pattern of GAP1 gene during the xiangmian18 different organs. The results showed that at the stage of gland formation in the seed germination, GAP1 gene expression at the gland development was stronger than gland no-development. The experiment showed that this gene was tissue specific. This gene was highly expressed in the vessel of stem cotton and phylogenetic of the leaves.5. Cloning and analysis on the representative pigment development related gene GhZIF.Based on the result of screening cotton microarray, the primers were designed according to DT463838. Using PCR to screen the constructed library, we got full length cDNA, named GhZIF (gland related protein 2, GenBank number: EU3703033) respectively.The complete nucleotide sequence of GhZIF cDNA has 1989 bp and contains a long open reading frame of 1464bp.The predicted protein sequence derived from the open reading frame produces a 487-amino acid polypeptide, with a calculated molecular mass of 53.75kD and a theoretical iso-electric point of 5.79; Protein subcellular location was predicted in the plasma membrane. The protein is hydrophobic property, which anchors membrane, containing a transporter domain.In situ hybridization was performed to analyze the expression pattern of GhZIF gene during the xiangmian18 different organs. The results showed that at the stage of gland formation in the seed germination, GhZIF gene expression at the gland no-development was stronger than gland development. This gene was highly expressed in the vessel of stem cotton,phylogenetic of the leaves and parenchyma cells.Therefore, the solid foundation had been laid for finding relevant genes and investigating their functions of cotton gland formation. It is significant to control the gland and the regulation and biosysthsis of gossypol at molecular level.更多还原