古巴柳叶橙叶形变异机理的初步研究

发布时间：2018-03-14 18:36

本文选题：芽变　切入点：叶片形态　出处：《西南大学》2017年硕士论文　论文类型：学位论文

【摘要】：芽变选种是柑橘重要的育种方式。叶是植物重要的营养器官,在遗传改良中占有举足轻重的地位,不同叶形状的变异可为优良新品系选择提供依据。研究柑橘叶片形态变异对解析叶发育机理、发掘重要的功能基因具有重要作用,还可为柑橘品种的遗传改良提供依据。本试验以古巴柳叶橙的窄叶野生型(N)和其宽叶突变体(W)为材料,利用形态学鉴定、显微切片技术对叶片形态变异组织结构进行鉴定,测定和比较了叶片发育相关的激素水平并利用转录组学和基因组重测序技术分别从生理和分子水平解析了古巴柳叶橙叶片变异的机理,主要结果如下:1、根据柑橘叶片发育的不同形态,将古巴柳叶橙野生型(N)及其突变体(W)叶片发育分为5个时期,分别是N1、N2、N3、N4、N5和W1、W2、W3、W4,W5。田间观察和形态学鉴定发现,叶片发育的各个时期,野生型和突变体叶片在叶宽、叶形指数、叶面积均有显著差异,进一步分析发现是叶片宽度的显著差异导致在叶形指数、叶面积上差异显著。通过嫁接繁殖后的叶片观察发现,宽叶型突变性状与嫁接前一致并且能稳定保持。2、对野生型N及其突变体W的叶片组织结构进行了石蜡切片、半薄切片和扫描电镜观察。石蜡切片观察发现N和W在叶发育的5个时期均有差异,主要表现在N的各个时期主叶脉的维管束组织占主脉比例较W的各个时期大。半薄切片观察发现,W5的叶片厚度比N5厚5.60%,差异显著;W5中构成栅栏组织的薄壁细胞排列疏松,而N5的栅栏组织排列更紧密;W5海绵组织细胞相对疏松,细胞间隙较多,而N5海绵组织细胞间隙相对较小。扫描电镜观察发现N5维管束结构、栅栏组织和海绵组织较W5紧密。3、采用HPLC-ESI-MS/MS法,测定了野生型和突变体叶片发育过程中的4种内源激素含量。结果显示,突变体中玉米素(ZT)的含量显著高于同时期的野生型且在二者中的变化趋势完全一致,即含量随着叶片的发育而降低;突变体中的脱落酸(ABA)含量也显著高于同时期的野生型,但变化趋势不同,在突变体中其含量随着叶片的发育而降低,而野生型中的变化趋势是先降低后升高;其含量在叶片发育中总体呈下降趋势;突变体中吲哚乙酸(IAA)含量比同时期的野生型低,但变化趋势不同,在突变体中其含量随着叶片的发育而降低,而野生型中的变化趋势是先降低后升高,在叶片发育过程中含量总体呈上升趋势;叶片发育过程中赤霉素3(GA3)含量较少,仅在W3与N3同时测出,表现为野生型高于突变体。4、将N1、N3、N5、W1、W3和W5时期叶片进行RNA-seq分析并以甜橙基因组(citrus.hzau.edu.cn)序列进行比对。结果显示,74.4%-78.7%的Clean Reads可以对比到基因组中,覆盖了全基因组的72.9-78.26%。6个样本总共组装拼接得到30921个基因。在野生型三个时期中,共有23728个表达基因,在突变体三个时期中,共有24089个表达基因。通过差异表达基因筛选发现,N1和W1时期的差异表达基因共有568个,N3和W3差异表达基因有207个,N5和W5差异表达基因有669个,其中在野生型和突变体叶片发育的所有时期均差异表达的基因有8个,分别是Cs1g16650、Cs1g26650、XLOC_005927、Cs3g17390、Cs6g08500、Cs7g27120、orange1.1t03155和orange1.1t02879。WEGO分析发现三个时期的差异表达基因在细胞组分中主要参与cell、cell part、membrane、membrane part等细胞组成;在分子功能中基因主要参与binding、catalytic、transcription regulator等功能;在生物学过程中主要参与biological regulation、cellular process、metabolic process、pigmentation等过程。随机选取9个基因进行荧光定量PCR验证,结果发现测序结果的FPKM值与qRT-PCR表达量趋势大部分吻合,说明测序结果真实可信。将在W和N叶片发育各个时期均差异表达的8个差异表达基因在5个时期进行qRT-PCR分析,发现其中有5个基因表达具有差异,且在叶片极性发育的第一个时期差异最为显著。5、将野生型N及其突变体W进行基因组重测序,均获得14.56G以上的原始数据量。通过生物信息学分析并与甜橙基因组进行对比,(http://citrus.hzau.edu.cn/orange/download/csi.chromosome.fa.tar.gz),发现两者均发生单核苷酸多态性(SNP)、小片段的插入和缺失(InDel)、结构变异(SV)和拷贝数变异(CNV),其中野生型N具有5390241个SNP,537847个InDel,23171个SV,6656个CNV;突变体W具有5410179个SNP,538048个InDel,24872个SV,6686个CNV。通过野生型和突变体样品间的比较,发现两者之间存在284495个SNP,5800个InDel,31552个SV和6679个CNV。对8个候选基因进行分析发现只有两个基因Cs7g27120和orange1.1t02879发生了SNP改变。本研究通过对古巴柳叶橙的野生型和宽叶突变体从性状的稳定性、形态特征、显微结构、激素水平和组学水平进行了初步的研究,初步发现宽叶突变性状能够稳定保持,在显微结构、激素水平等有显著差异,并发现了8个与性状相关的重要候选基因和在基因组水平上的差异,为进一步明晰叶片形态变异的机理奠定了坚实的基础。
[Abstract]:Is an important way of sport selection breeding. Citrus leaf is an important organ of plant nutrition, plays an important role in genetic improvement and variation of different leaf shape and can provide the basis for selection of fine lines. Study on the mechanism of citrus leaf morphological variation of leaf development, plays an important role in exploring an important functional gene, provide the basis for genetic improvement also for citrus cultivars. In this experiment, the wild type leaves Cuba willow orange (N) and the wide leaf mutant (W) as material, using morphological identification, microscopic technique on leaf morphological variation structure were identified, measured and compared the leaf growth hormone level and the use of transcriptomics and genome sequencing technology from the physiological and molecular level analysis of the mechanism of Cuba willow orange leaf variation. The main results are as follows: 1, according to the different forms of citrus leaf development, the ancient Pakistan willow orange wild type (N) and its mutant (W) leaf development is divided into 5 periods, namely N1, N2, N3, N4, N5 and W1, W2, W3, W4, W5. field observation and morphological identification, each period of leaf development, wild type and mutant leaves in broad leaves the leaf number, leaf area had significant differences, further analysis found significant differences in leaf width, leaf index, leaf area differences. Through the observation of the leaf grafting found broad leaf type traits and.2 mutation remain stable and consistent before grafting, leaf tissue structure of wild type N and its mutant W for the paraffin section and semi thin sections and scanning electron microscope. The paraffin section observation showed that N and W were in the 5 period differences in leaf development, mainly in the various periods of N main vein vascular bundle tissue for each period of the main vein proportion is W. Semi thin section observation Now, the thickness of leaf W5 thicker than the N5 5.60%, the difference was significant; W5 composed of palisade tissue parenchyma cells arranged loosely, and palisade tissue N5 arranged more closely; relatively loose W5 sponge cells, cell gap is more, while the N5 sponge tissue cell gap is relatively small. SEM observation showed that the N5 vascular structure, palisade tissue spongy tissue than W5.3 closely, using the HPLC-ESI-MS/MS method, 4 kinds of endogenous hormone content of wild-type and mutant leaves were determined. The results showed that the mutant zeatin (ZT) content was significantly higher than that of the wild type at the same time and the change of the two exactly the same trend, namely content decreased with the development of leaves; abscisic acid of the mutant (ABA) were significantly higher than those of the wild type at the same time period, but the change trend is different, the content in the mutant with the development of leaves decreased, while the wild type of change The trend is first decreased and then increased; the content in the leaves decreased; indole acetic acid (IAA) content in the mutant than the wild type in the same period, but the trend is different, the content in the mutant with the development of leaves decreased, and the change trend of wild type is decreased at first and then increased. In the process of leaf development in the overall upward trend during leaf development 3; gibberellin (GA3) content is less, only in the W3 and N3 measured at the same time, performance is higher than that of the wild type of mutant.4, N1, N3, N5, W1, W3 and W5 in leaves were analyzed by RNA-seq and the orange genome (citrus.hzau.edu.cn) sequence for comparison. The results showed that 74.4%-78.7% Clean Reads can be compared to the genome, covering the whole genome of 72.9-78.26%.6 samples from a total of 30921 genes are assembled in the wild type. The three period, a total of 23728 genes Because, in the mutant in the three period, a total of 24089 genes. Genes found by differential expression between N1 and W1 in the expression of 568 genes were N3 and W3, there were 207 differentially expressed genes, N5 and W5, there were 669 differentially expressed genes, the expression of difference in all phases of the wild type and mutants of leaf development and 8 genes, respectively Cs1g16650, Cs1g26650, XLOC_005927, Cs3g17390, Cs6g08500, Cs7g27120, orange1.1t03155 and orange1.1t02879.WEGO analysis showed that the differences between the three periods of gene expression in cell components in the main parameters and cell, cell part, membrane membrane, composed of part cells; the molecular function of gene mainly involved in binding, catalytic, transcription, regulator and other functions; in the biological process is mainly involved in the biological regulation, cellular process, metabolic process, pigmentation and other randomly selected process. The 9 genes were verified by real time PCR, the sequencing results of FPKM value trend consistent with most of the expression of qRT-PCR, indicating the sequencing result is true and credible. The 5 period gene in qRT-PCR analysis of 8 differentially expressed the different developmental stages in W and N leaves were differentially expressed, found that there are 5 genes the expression is different, and in the first period of leaf polarity development. The most significant.5, wild type N and its mutant W genome sequencing, were more than 14.56G. The original data by bioinformatics analysis and orange gene groups were compared (http://citrus.hzau.edu.cn/orange/download/csi.chromosome.fa.tar.gz), found that the two were single nucleotide polymorphism (SNP), insertion and deletion of small fragments (InDel), structural variation (SV) and copy number variation (CNV), the wild type N with 5390241 SNP, 5378 47 InDel, 23171 SV, 6656 CNV; mutant W with 5410179 SNP, 538048 InDel, 24872 SV, 6686 CNV. by wild type and mutant samples, it is found that there are 284495 SNP between the two, 5800 InDel, 31552 SV and 6679 CNV. of 8 candidate base the analysis found that only two genes Cs7g27120 and orange1.1t02879 had SNP change. Through the study of wild type of Cuba willow and wide leaf mutant from orange traits stability, morphology, microstructure, hormone level and group level has conducted the preliminary research, preliminary found wide leaf mutant traits could be maintained in. The microstructure, hormone levels have significant differences, and found the difference between the 8 important candidate genes associated with the traits and at the genomic level, and laid a solid foundation for further clarifying the mechanism of leaf morphological variation.

【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S666.4

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