發(fā)布時(shí)間：2018-03-14 18:36

  本文選題：芽變　切入點(diǎn)：葉片形態(tài)　出處：《西南大學(xué)》2017年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：芽變選種是柑橘重要的育種方式。葉是植物重要的營(yíng)養(yǎng)器官,在遺傳改良中占有舉足輕重的地位,不同葉形狀的變異可為優(yōu)良新品系選擇提供依據(jù)。研究柑橘葉片形態(tài)變異對(duì)解析葉發(fā)育機(jī)理、發(fā)掘重要的功能基因具有重要作用,還可為柑橘品種的遺傳改良提供依據(jù)。本試驗(yàn)以古巴柳葉橙的窄葉野生型(N)和其寬葉突變體(W)為材料,利用形態(tài)學(xué)鑒定、顯微切片技術(shù)對(duì)葉片形態(tài)變異組織結(jié)構(gòu)進(jìn)行鑒定,測(cè)定和比較了葉片發(fā)育相關(guān)的激素水平并利用轉(zhuǎn)錄組學(xué)和基因組重測(cè)序技術(shù)分別從生理和分子水平解析了古巴柳葉橙葉片變異的機(jī)理,主要結(jié)果如下:1、根據(jù)柑橘葉片發(fā)育的不同形態(tài),將古巴柳葉橙野生型(N)及其突變體(W)葉片發(fā)育分為5個(gè)時(shí)期,分別是N1、N2、N3、N4、N5和W1、W2、W3、W4,W5。田間觀察和形態(tài)學(xué)鑒定發(fā)現(xiàn),葉片發(fā)育的各個(gè)時(shí)期,野生型和突變體葉片在葉寬、葉形指數(shù)、葉面積均有顯著差異,進(jìn)一步分析發(fā)現(xiàn)是葉片寬度的顯著差異導(dǎo)致在葉形指數(shù)、葉面積上差異顯著。通過(guò)嫁接繁殖后的葉片觀察發(fā)現(xiàn),寬葉型突變性狀與嫁接前一致并且能穩(wěn)定保持。2、對(duì)野生型N及其突變體W的葉片組織結(jié)構(gòu)進(jìn)行了石蠟切片、半薄切片和掃描電鏡觀察。石蠟切片觀察發(fā)現(xiàn)N和W在葉發(fā)育的5個(gè)時(shí)期均有差異,主要表現(xiàn)在N的各個(gè)時(shí)期主葉脈的維管束組織占主脈比例較W的各個(gè)時(shí)期大。半薄切片觀察發(fā)現(xiàn),W5的葉片厚度比N5厚5.60%,差異顯著;W5中構(gòu)成柵欄組織的薄壁細(xì)胞排列疏松,而N5的柵欄組織排列更緊密;W5海綿組織細(xì)胞相對(duì)疏松,細(xì)胞間隙較多,而N5海綿組織細(xì)胞間隙相對(duì)較小。掃描電鏡觀察發(fā)現(xiàn)N5維管束結(jié)構(gòu)、柵欄組織和海綿組織較W5緊密。3、采用HPLC-ESI-MS/MS法,測(cè)定了野生型和突變體葉片發(fā)育過(guò)程中的4種內(nèi)源激素含量。結(jié)果顯示,突變體中玉米素(ZT)的含量顯著高于同時(shí)期的野生型且在二者中的變化趨勢(shì)完全一致,即含量隨著葉片的發(fā)育而降低;突變體中的脫落酸(ABA)含量也顯著高于同時(shí)期的野生型,但變化趨勢(shì)不同,在突變體中其含量隨著葉片的發(fā)育而降低,而野生型中的變化趨勢(shì)是先降低后升高;其含量在葉片發(fā)育中總體呈下降趨勢(shì);突變體中吲哚乙酸(IAA)含量比同時(shí)期的野生型低,但變化趨勢(shì)不同,在突變體中其含量隨著葉片的發(fā)育而降低,而野生型中的變化趨勢(shì)是先降低后升高,在葉片發(fā)育過(guò)程中含量總體呈上升趨勢(shì);葉片發(fā)育過(guò)程中赤霉素3(GA3)含量較少,僅在W3與N3同時(shí)測(cè)出,表現(xiàn)為野生型高于突變體。4、將N1、N3、N5、W1、W3和W5時(shí)期葉片進(jìn)行RNA-seq分析并以甜橙基因組(citrus.hzau.edu.cn)序列進(jìn)行比對(duì)。結(jié)果顯示,74.4%-78.7%的Clean Reads可以對(duì)比到基因組中,覆蓋了全基因組的72.9-78.26%。6個(gè)樣本總共組裝拼接得到30921個(gè)基因。在野生型三個(gè)時(shí)期中,共有23728個(gè)表達(dá)基因,在突變體三個(gè)時(shí)期中,共有24089個(gè)表達(dá)基因。通過(guò)差異表達(dá)基因篩選發(fā)現(xiàn),N1和W1時(shí)期的差異表達(dá)基因共有568個(gè),N3和W3差異表達(dá)基因有207個(gè),N5和W5差異表達(dá)基因有669個(gè),其中在野生型和突變體葉片發(fā)育的所有時(shí)期均差異表達(dá)的基因有8個(gè),分別是Cs1g16650、Cs1g26650、XLOC_005927、Cs3g17390、Cs6g08500、Cs7g27120、orange1.1t03155和orange1.1t02879。WEGO分析發(fā)現(xiàn)三個(gè)時(shí)期的差異表達(dá)基因在細(xì)胞組分中主要參與cell、cell part、membrane、membrane part等細(xì)胞組成;在分子功能中基因主要參與binding、catalytic、transcription regulator等功能;在生物學(xué)過(guò)程中主要參與biological regulation、cellular process、metabolic process、pigmentation等過(guò)程。隨機(jī)選取9個(gè)基因進(jìn)行熒光定量PCR驗(yàn)證,結(jié)果發(fā)現(xiàn)測(cè)序結(jié)果的FPKM值與qRT-PCR表達(dá)量趨勢(shì)大部分吻合,說(shuō)明測(cè)序結(jié)果真實(shí)可信。將在W和N葉片發(fā)育各個(gè)時(shí)期均差異表達(dá)的8個(gè)差異表達(dá)基因在5個(gè)時(shí)期進(jìn)行qRT-PCR分析,發(fā)現(xiàn)其中有5個(gè)基因表達(dá)具有差異,且在葉片極性發(fā)育的第一個(gè)時(shí)期差異最為顯著。5、將野生型N及其突變體W進(jìn)行基因組重測(cè)序,均獲得14.56G以上的原始數(shù)據(jù)量。通過(guò)生物信息學(xué)分析并與甜橙基因組進(jìn)行對(duì)比,(http://citrus.hzau.edu.cn/orange/download/csi.chromosome.fa.tar.gz),發(fā)現(xiàn)兩者均發(fā)生單核苷酸多態(tài)性(SNP)、小片段的插入和缺失(InDel)、結(jié)構(gòu)變異(SV)和拷貝數(shù)變異(CNV),其中野生型N具有5390241個(gè)SNP,537847個(gè)InDel,23171個(gè)SV,6656個(gè)CNV;突變體W具有5410179個(gè)SNP,538048個(gè)InDel,24872個(gè)SV,6686個(gè)CNV。通過(guò)野生型和突變體樣品間的比較,發(fā)現(xiàn)兩者之間存在284495個(gè)SNP,5800個(gè)InDel,31552個(gè)SV和6679個(gè)CNV。對(duì)8個(gè)候選基因進(jìn)行分析發(fā)現(xiàn)只有兩個(gè)基因Cs7g27120和orange1.1t02879發(fā)生了SNP改變。本研究通過(guò)對(duì)古巴柳葉橙的野生型和寬葉突變體從性狀的穩(wěn)定性、形態(tài)特征、顯微結(jié)構(gòu)、激素水平和組學(xué)水平進(jìn)行了初步的研究,初步發(fā)現(xiàn)寬葉突變性狀能夠穩(wěn)定保持,在顯微結(jié)構(gòu)、激素水平等有顯著差異,并發(fā)現(xiàn)了8個(gè)與性狀相關(guān)的重要候選基因和在基因組水平上的差異,為進(jìn)一步明晰葉片形態(tài)變異的機(jī)理奠定了堅(jiān)實(shí)的基礎(chǔ)。
[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.

【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：S666.4

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