本文選題：番茄　切入點(diǎn)：葉色突變體　出處：《華中農(nóng)業(yè)大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：番茄是重要的蔬菜作物,植株光合效率對番茄的產(chǎn)量及品質(zhì)有著極其重要的影響,而葉片是其光合積累有機(jī)物的場所。葉色突變是比較常見并容易觀察表型的一類突變體,該類突變體的形成是由于葉綠素含量、葉綠體發(fā)育以及光合作用等過程受到影響導(dǎo)致,從而影響作物品質(zhì)或產(chǎn)量。本研究以在遺傳轉(zhuǎn)化過程發(fā)現(xiàn)的一株番茄雜色葉突變體vg為研究對象,通過從生理、遺傳、及分子水平對突變體進(jìn)行表型鑒定、突變原因、遺傳分析、基因定位、功能驗(yàn)證等研究,了解vg突變體雜色葉產(chǎn)生的機(jī)制,為研究番茄光合途徑及效率提供理論基礎(chǔ)。主要的研究結(jié)果如下:1.番茄葉色突變體vg的表型:vg突變體表現(xiàn)為苗期正常,在播種后一個(gè)月左右開始出現(xiàn)葉色變化,首先是剛成熟的葉片出現(xiàn)部分泛白,隨著幼苗生長有泛白的葉片增多,并且出現(xiàn)包括生長點(diǎn)的幼葉出現(xiàn)黃化,植株長勢受到抑制。秋季育苗高溫強(qiáng)光照條件下,表型更加明顯,生長勢更弱。2.番茄葉色突變體vg的生理特性分析:觀察葉綠體的超微結(jié)構(gòu)發(fā)現(xiàn)與AC相比,vg突變體黃化葉片的葉綠體形態(tài)異常,沒有類囊體片層形成;vg突變體泛白葉片和正常綠色葉片的葉綠體形態(tài)基本正常,但泛白葉片葉綠體的類囊體片層的排列分散、類囊體基粒較少;正常綠色葉片葉綠體的類囊體片層排列不緊密。且在vg突變體的葉片中,沒有明顯可見的淀粉粒形成。分別測定三種葉色的葉綠素含量發(fā)現(xiàn),黃化葉片的葉綠素a、葉綠素b都顯著降低,泛白葉片的葉綠素b降低,正常綠色葉片的葉綠素含量基本不變。且正常綠色葉片的可溶性糖含量以及淀粉含量都明顯減少。3.番茄葉色突變體vg的共分離及遺傳分析表明:vg突變體的表型與轉(zhuǎn)入的基因沒有關(guān)系,且不是T-DNA插入導(dǎo)致的突變,而是遺傳轉(zhuǎn)化過程引起的突變。以LA1589為母本,vg突變體為父本構(gòu)建F_2遺傳分離群體,分析表明vg突變體的突變表型是單核基因控制的隱性性狀。4.利用分離群體基因定位:從F_2群體分離的45株有突變表型的單株,以及在每條染色體上開發(fā)并篩選出具有穩(wěn)定多態(tài)性的2個(gè)In Del標(biāo)記,將目的基因定位在第7條染色體。進(jìn)一步開發(fā)篩選了15對In Del分子標(biāo)記,結(jié)果將目的基因定位在7-59980和7-60636兩個(gè)標(biāo)記之間物理距離為756 kb的區(qū)段。擴(kuò)大分離F_2群體,分離出了182株有突變表型的單株用于精細(xì)定位,利用1個(gè)In Del和8個(gè)CAPS分子標(biāo)記,將目的基因定位在s6027和Ba6040兩個(gè)標(biāo)記之間物理距離為128 kb的區(qū)段。5.基因侯選:利用預(yù)測網(wǎng)站和基因序列分析,分析可知在目的區(qū)段有21個(gè)開放閱讀框(ORFs)。其中ORF10、ORF11、ORF12和ORF13與葉綠體發(fā)育或葉綠素合成相關(guān),而已有研究表明ORF10的同源基因在擬南芥中導(dǎo)致類囊體形成受到阻礙。在突變體和AC中擴(kuò)增4個(gè)基因的編碼序列以及ORF10的啟動子序列,均未發(fā)現(xiàn)堿基序列上的差異。6.基因時(shí)空表達(dá)分析:利用q RT-PCR分析除傷誘導(dǎo)相關(guān)基因外的12個(gè)基因在AC和突變體中的表達(dá)量。結(jié)果顯示ORF9、ORF10和ORF13的表達(dá)量顯著降低,ORF11的表達(dá)量表現(xiàn)顯著升高。而ORF9在ORF10的啟動子上,ORF10編碼類囊體形成蛋白。因此,推測ORF10可能是導(dǎo)致突變表型的目的基因。7.利用VIGS技術(shù)驗(yàn)證候選基因功能:構(gòu)建了ORF10和ORF13的p TRV2病毒表達(dá)載體,以AC為浸染受體。結(jié)果顯示,AC沉默ORF13不會產(chǎn)生突變表型,沉默ORF10使AC表現(xiàn)與突變表型相似的表型。因此,確定ORF10是導(dǎo)致突變表型的目的基因。
[Abstract]:Tomato is an important vegetable crop, has a very important effect on plant photosynthetic efficiency on Tomato Yield and quality, and the leaf photosynthetic organic matter accumulation in place. Leaf color mutation is a relatively common and easy to observe the mutant phenotype, the formation of this type of mutant is due to the chlorophyll content, chloroplast development and the photosynthesis process affected, thus affecting the quality of crops or production. In this study, a strain of tomato genetic transformation process found in variegated leaf mutant VG as the research object, from the physiological, genetic, and molecular phenotype identification of mutants, genetic analysis, gene mapping, research on functional verification. To understand the mechanism of VG mutant variegated leaves, provide a theoretical basis for the study of tomato photosynthetic pathway and efficiency. The main results are as follows: 1. the phenotype of tomato leaf color mutant VG: VG mutation The body is normal seedling after sowing, in a month or so began to leaf color changes, the first is just part of mature leaves appear white, with white leaf seedling growth have increased, and including the growing point of leaf etiolation, plant growth was inhibited. The autumn Ji Yumiao high temperature high light conditions, the phenotype is more obvious analysis of physiological characteristics, the growth potential of weaker.2. tomato leaf color mutant VG: the ultrastructure of chloroplasts found that compared with AC, the abnormal chloroplast morphology of VG mutant leaves, no thylakoid lamella formation; chloroplast morphology of VG mutant white leaf and green leaf basically normal, but the chloroplast thylakoids of white leaf the scattered arrangement, the grana lamella is less; the normal arrangement of green leaf chloroplast was not close. And in the leaves of VG mutants, not visible The starch grains formed. Chlorophyll content of three leaf color were measured, yellow leaf chlorophyll a, chlorophyll b decreased significantly, white leaf chlorophyll b decreased, chlorophyll content of normal green is basically the same. And the content of soluble sugar in leaves of normal green and starch content were significantly reduced in total isolation and genetic analysis.3. tomato leaf color mutant VG showed that Never mind VG mutant phenotype and gene, and not the T-DNA insertion mutations resulting in mutation, but genetic transformation process. By taking LA1589 as female parent and VG as male parent mutant F_2 segregation population construction, analysis suggested that the mutant phenotype of VG mutant gene mapping using isolated populations.4. is a single recessive nuclear gene control: isolated from F_2 group with single mutant phenotypes of 45 strains, and in each chromosome and screened with stable development 2 In Del marked state of the target gene, located in chromosome seventh. Further screening 15 pairs of In Del molecular marker, gene localization results will be in the physical distance between 7-59980 and 7-60636 of two markers was 756 KB. The section expanded separation of F_2 groups, 182 strains were isolated from the plants with mutant phenotype for fine mapping, using 1 In Del and 8 CAPS markers, the target gene was mapped between s6027 and Ba6040 two marks the physical distance of 128 KB candidate region of the.5. gene using predictive analysis and site selection: gene sequence analysis shows that there are 21 open reading frames in the head section (ORFs) the ORF10, ORF11, ORF12 and ORF13 and chloroplast development or chlorophyll synthesis related, it has been reported that ORF10 homologous gene in Arabidopsis led to the formation of thylakoids is hindered. Amplification of 4 genes encoding sequence in the mutant and AC ORF10 and the promoter sequences were not found differential expression analysis of.6. gene sequence on time: the use of Q RT-PCR analysis in the expression of 12 genes induced by injury related genes outside the AC and in mutants. The results showed that the ORF9 expression of ORF10 and ORF13 were significantly decreased and the expression of ORF11 increased significantly while ORF9 in the ORF10 promoter, ORF10 encoding thylakoid formation protein. Therefore, we speculated that ORF10 may lead to.7. gene mutation phenotype using validation of candidate gene VIGS Technology: Construction of ORF10 and ORF13 P TRV2 virus expression vector with AC disseminated receptor. The results showed that AC silencing ORF13 does not produce mutations the silencing of ORF10 phenotype, AC and mutant phenotype similar phenotypes. Therefore, ORF10 is the result of gene mutation phenotype.

【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S641.2;Q943.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前3條

1 曹莉;王輝;孫道杰;馮毅;;小麥黃化突變體光合作用及葉綠素?zé)晒馓匦匝芯縖J];西北植物學(xué)報(bào);2006年10期

2 趙琦,唐崇欽,匡廷云;玉米突變體(zb/zb)的葉綠體光合特性[J];Acta Botanica Sinica;1997年11期

3 錢前,朱旭東,曾大力,張小惠,嚴(yán)學(xué)強(qiáng),熊振民;細(xì)胞質(zhì)基因控制的新特異材料白綠苗的研究[J];作物品種資源;1996年04期

相關(guān)碩士學(xué)位論文 前4條

1 高文婧;番茄葉片白化轉(zhuǎn)綠控制基因wv的精細(xì)定位[D];華中農(nóng)業(yè)大學(xué);2016年

2 楊明澤;番茄ShWRKY6基因的抗逆功能研究[D];華中農(nóng)業(yè)大學(xué);2015年

3 王新華;水稻白條紋葉突變體B38的表型分析及突變基因的精細(xì)定位[D];南京農(nóng)業(yè)大學(xué);2013年

4 郭明;番茄葉色黃化突變體黃化性狀分子標(biāo)記及蛋白變化研究[D];東北農(nóng)業(yè)大學(xué);2010年

，

本文編號：1612510