【作者】 李廷春；

【导师】 林毅；

【作者基本信息】 安徽农业大学 ， 作物遗传育种， 2011， 博士

【摘要】 棕色棉是一种纤维呈现天然棕色的彩色棉,由于其本身带有天然的颜色,在棉花纺织生产过程中,可以免去漂白、消毒、染色等过程,污染少、环保健康。但是,棕色棉与白色棉相比,品质不够理想,存在颜色不够纯正、色素分布不均匀、色素遗传不稳定等缺陷,严重制约了棕色棉的生产与应用。前人从棕色棉纤维中克隆了原花色素合途径中的结构基因(F3H, DFR, CHI等),结合棕色棉纤维色素化学性质的鉴定结果,初步推断棕色棉纤维色素的合成前体为缩合单宁,即原花色素。目前对棕色棉纤维发育过程中原花色素合成、积累规律的研究较少,关于白色棉与棕色棉中原花色素合成、积累的差异鲜见报道,关于棕色棉纤维色素与原花色素的关系仍有待深入研究。棕色棉的纤维颜色形成于纤维发育的中后期阶段,研究表明,纤维颜色的呈现与光照有关,但关于纤维色素合成过程中是否存在光质调控尚不清楚,纤维色素合成和积累与光是否存在直接关系仍需进一步研究。本课题以棕色棉为材料,通过对纤维组织中原花色素的染色分析、含量测定及其结构基因表达分析等,研究棕色棉纤维色素合成与原花色素的关系,并结合MSAP与cDNA-AFLP技术,研究棕色棉纤维发育过程中的DNA甲基化模式变化与基因表达差异,分析棕色棉纤维发育的分子机制。同时,通过采用不同透光膜处理棕色棉幼苗,研究不同光质对棕色棉幼苗中原花色素合成、积累的影响,探讨光质调控原花色素合成的生理与分子机制,为进一步明确原花色素与纤维色素的关系,解决棕色棉纤维色素颜色不纯正、色素分布不均匀的缺陷提供理论依据。主要研究结果如下：1.以棕色棉和白色棉为材料,采用DMACA和TBO对不同发育阶段纤维组织进行化学染色,并对纤维发育过程中,纤维组织中的原花色素含量变化与结构基因GhCHS、GhF3H、GhDFR、GhANS与GhANR表达进行分析,结果表明,棕色棉和白色棉纤维中原花色素的合成起始于纤维细胞的突起阶段,但白色棉纤维中原花色素含量低,并在5DPA后开始降低,20DPA时就检测不到原花色素；而在棕色棉纤维发育的5-40DPA时期,纤维中原花色素含量较高,呈先升后降的趋势,在15DPA原花色素含量最高,并且,在棕色棉纤维中,原花色素合成的结构基因GhCHS、GhF3H、 GhDFR、GhANS与GhANR只在纤维发育的早、中期表达,在花后10天、15天表达量最高,花后15天后表达减弱,纤维生长发育后期表达可能较低。2.利用MSAP与cDNA-AFLP技术研究棕色棉纤维发育过程中(5DPA-25DPA)的DNA甲基化模式变化与基因表达差异。66对MSAP选扩引物每个样品平均共扩增出1010.5个带型,每对引物扩增出11-27个片段,平均15.31个片段。随着纤维发育进程的推进,纤维DNA甲基化条带总数、甲基化比率、全甲基化比率逐渐升高,纤维发育的过程中,DNA发生甲基化的位点数逐渐增多。采用64对cDNA-AFLP的引物组合,每对引物组合扩增的总条带数在32-51条之间,平均39.8条,其中,有75条转录衍生片段呈多态性。通过对30条多态性转录衍生片段的克隆、测序与同源性分析发现,30条序列中与已知的棉花相关序列同源性较高的序列较多,共有19条来自于棉属植物。而根据30条转录衍生片段的Blast比对结果,有13条转录衍生片段和报道的已知功能的基因同源,其余17条转录衍生片段的功能未知。3.采用红、黄、蓝、白四种透光膜处理棕色棉幼苗,研究光质对棕色棉原花色素、表型性状及其光合系统的影响。结果表明,红膜处理能提高棉花幼苗各组织中原花色素含量,黄膜与蓝膜处理对原花色素合成、积累有抑制作用；红膜、黄膜处理对植物的生长有促进作用、蓝膜处理可促进棉花幼苗根系生长,但会抑制植株生长；而经黄膜、蓝膜与红膜处理后,棕色棉幼苗光能的利用效率降低,净光合速率也发生明显下降,光合产物积累减少,但白膜处理的棕色棉植株仍能较好的吸收、转换、利用光能,并能保持较高的净光合速率。4.利用MSAP皮术研究不同透光膜处理对棕色棉幼苗DNA甲基化模式和水平变化的影响,探讨光质对棕色棉生长、发育影响的表观遗传机制。选取了66对MSAP选扩引物进行PCR扩增反应,每个样品平均共扩增出1300.5个带型,每对引物扩增出15-32个片段,平均19.7个片段。经不同膜处理后,红膜、黄膜和蓝膜处理均增加了棉花DNA总甲基和全甲基化的比率,但蓝膜处理降低了棉花DNA半甲基化比例。对甲基化多态性片段的测序、分析表明,DNA甲基化发生改变的位点既存在于基因组的编码区,也存在于非编码区；棉花幼苗中丙酮酸激酶同源基因Seql与水通道蛋白同源基因Seq4的表达受光质调控,基因的激活表达与其甲基化位点的去甲基化有关。更多还原

【Abstract】 Brown cotton is a kind of naturally colored cotton, which accumulates brown pigment in fiber. Because of less processing and dying procedures, it could induce less harmful chemical effects and is friendlier to environment than white cotton. However, in contrast with white cotton, brown cotton has many defects, such as mixed color, inhomogeneous distribution and unstable inheritance of pigments, which could limit its use and popularization.In previous studies, several flavonoid structural genes including encoding dihydroflavonol reductase (DFR), anthocyanidin synthase (ANS), flavanone3-hydroxylase (F3H), and anthocyanidin reductase (ANR) were cloned from brown fiber. The analysis of chemical properties of extracted compounds primarily demonstrated that the pigment in brown fiber might be proanthocyanidins (PAs, also called condensed tannins). However, the reports on synthesis and accumulation rules of PAs are less and the relationship between PAs and brown pigment in fiber are not yet clear by now.For brown cotton, during the middle and late development stages of fiber, the brown pigment was accumulated in fiber. In the accumulated procedure, light might be related to the sun light irradiation. However, it needs to further research whether the light has effects on pigment synthesis or not.In the experiment, on the one hand, the relationship between brown pigment and PAs was studied. On the another hand, the variation of DNA methylation and gene expression were analyzed by using MSAP and cDNA-AFLP methods during the development stages from5to25DPA. On the third hand, the seedlings of brown cotton were treated with different color films, and the effects of light quality on PAs synthesis were discussed. The results of the experiment were as follows:1. A brown fiber line and a white fiber cultivar were selected to determine the factors that affect the pigmentation in brown fiber. Accordingly, the fibers in different developmental stages were collected to verify the presence of PAs by p-dimethylami-nocinnamaldehyde (DMACA) and toluidine blue O (TBO) staining. The PAs content and related gene expressions were determined. As a result, PAs synthesis started at the stage of papillary epidermic cells of seeds and there were obvious differences on the aspect of PAs synthesis in fiber between white cotton and brown cotton. For white fiber, the PAs content reached maximum at5DPA stage, and then gradually decreased to zero. But for brown fiber, the PAs content was increased from5to15DPA stage, and reached the maximum at the15DPA stage, then gradually decreased from15to40DPA stage. In brown cotton, PAs synthesis continued for the whole developmental stage from5to40DPA. PAs content in brown fiber was far more than that in white fiber, which may be the reason why the brown pigment accumulated in brown fiber.2. Brown fibers were collected to analyze the variation of DNA methylation and gene expression by using MSAP and cDNA-AFLP methods during the development stages from5to25DPA. The results showed that by MSAP analysis, a total of1010.5bands per samples and11-27bands per primer combination were amplified by using66pairs of primer combinations. During the development process of fiber, the sum of methylated sites, the ratio of methylated sites and percentage of fully methylated sites were gradually increased with the advancement of fiber development stages. However, by cDNA-AFLP analysis, a total of32-51bands with average of39.8bands per primer combination were amplified by using64pairs of primer combinations. There are75polymorphic transcription derived fragments (TDFs) were detected, and among that,30TDFs were sequenced and analyzed. By blasting at NCBI website,19TDFs were detected to be homologous with genes from cotton. According to blasting results, among30TDFs,13TDFs were homologous with known functional genes; however,17TDFs were homologous with unknown function genes.3. The seedlings of brown cotton were treated with different color films, and the effects of light quality on PAs synthesis, phenotype traits and photosynthetic system were studied. As a result, red film treatment increased the PAs content, however, yellow and blue films treatment inhibited the PAs synthesis. After treatment with different color films, plant growth were accelerated by red and yellow films, root growth was promoted by blue film. The Physiological Parameters such as light use efficiency, net photosynthetic rate and accumulation of photosynthetic products were decreased after the treatment with red, blue and yellow films. The plants treated with white films maintained better light use efficiency and higher net photosynthetic rate.4. The effects of light quality on variation of DNA methylation were analyzed using the seedlings of brown cotton treated with different color films. The results showed that a total of1300.5bands per samples and15-32bands with an average of19.7bands per primer combination were amplified by using66pairs of primer combinations. The ratios of total methylated sites and fully-methylated sites were increased after treatment with red, yellow and blue films, moreover, blue film treatment decreased the percentage of hemi-methylated sites. In addition,10polymorphic fragments were sequenced and cloned. By blasting at NCBI, the sequences alignment revealed that both coding and non-coding regions could be methylated or demethylated by different light quality. The expression of seq1(homologous with pyruvate kinase) and seq2(homologous with MIP family) were regulated by light quality, which were closely related to the procedure of the demethylation of methylated sites for the genes during the activated expression.更多还原