【作者】 卓智勇；

【导师】 张志胜；

【作者基本信息】 华南农业大学 ， 风景园林硕士（专业学位）， 2020， 硕士

【摘要】 兰花（Cymbidium）具有重要的观赏、文化和经济价值以及广阔的市场前景。重离子辐照育种具有操作简单、诱变率高、培育周期短等优点,在花卉育种研究中已有一些研究报道,但在兰花上报道较少。本研究以杂交组合17-33、‘12g-5’、‘小凤兰’根状茎及‘宫粉佳人兰’类原球茎为材料,研究重离子辐照对兰花中间繁殖体存活、分化和诱变的效应,寻找在‘君豪兰’根茎叶中进行基因表达分析的内参基因,对‘线艺君豪兰’和‘君豪兰’进行转录组测序和比较分析,找出与线艺性状突变有关的候选基因。主要研究结果如下:1.兰花中间繁殖体的重离子辐照效应。重离子辐照导致杂交组合17-33、‘12g-5’、‘小凤兰’根状茎及‘宫粉佳人兰’类原球茎死亡,40 Gy辐照的最终死亡率分别为100%、96.15%、92.00%和95.19%。20 Gy的17-33根状茎和40 Gy的‘宫粉佳人兰’类原球茎分别在培养160 d和240 d后恢复生长。SSR标记分析结果表明,0 Gy的17-33根状茎和0、40 Gy的‘宫粉佳人兰’类原球茎均未扩增出多态性条带,而20 Gy的17-33根状茎扩增出两条多态性条带,多态率为2.0%。2.利用ge Norm、Norm Finder和Best Keeper软件对Rps3、GAPDH、ACT、EF1α、Ch ACT、Ch TUB、Ch UBQ、Ch EF-1a、Chrpo B、Rps19、18S、Rpo B、TUB、26S和Psa B15个内参基因进行了表达稳定性评估。结果表明,Rps19和Ch UBQ适合作为‘君豪兰’根茎叶中基因表达分析的内参基因。3.利用Illumina Hiseq X技术对‘君豪兰’和‘线艺君豪兰’的叶片和根、茎混样进行转录组测序,共获得75,985个unigene,其中差异表达基因有13059个,在差异表达基因中,4034个基因表达上调,9025个基因表达下调。在‘君豪兰’和‘线艺君豪兰’叶片中差异表达基因有8808个,2415个基因表达上调,6393个基因表达下调。KEGG分析结果显示,叶片中差异表达基因富集在次生代谢产物的生物合成等124个生物代谢通路中,其中,与叶艺发生有关的通路有花青素苷合成、光合作用-天线蛋白、卟啉与叶绿素代谢、类黄酮生物合成和光合作用途径,在这些代谢通路中差异表达基因数分别为8、7、3、16和14个。4.从与叶艺发生有关的生物代谢通路中选择20个差异表达基因进行q RT-PCR分析。结果筛选出2个与兰花线艺性状突变有关的候选基因,分别为CYP76B10和PSBO。通过上述研究,明确了¹²C⁶⁺重离子辐照对兰花中间繁殖体存活和诱变效应,初步确定了与君豪兰线艺突变的相关基因,为深入研究重离子辐照诱变的分子机理、建立兰花高效重离子辐照诱变育种技术体系和选育突破性兰花新品种奠定了基础。更多还原

【Abstract】 Cymbidium is a traditional Chinese orchid with important ornamental,cultural and economic value and broad market prospect.Heavy ion irradiation breeding has the advantages of simple operation,high mutagenesis rate,short breeding period,which had been widely used in ornamental plant breeding.However,there were only a few reports focused on orchid.In this study,the rhizomes of Cymbidium 17-33,C.‘12g-5’,C.‘Xiao Feng’and protocorm like-bodies（PLBs）of C.‘Gong Fen Jia Ren’were used as test materials to investigate the effect of heavy ion radiation on survival,differentiation and induced mutation of intermediate propagules.C.‘Jun Hao’was employed to explore internal reference genes for gene expression analysis in root,pseudobulb and leaf,and to find candidate genes related to thread art mutation on leaf by using of transcriptome sequencing and q RT-PCR methods.The main findings are as follows:1.The effect of heavy ion irradiation on survival,differentiation and mutation of intermediate propagules in Cymbidium.Heavy ion irradiation caused the death of rhizomes of C.17-33,C.‘12g-5’,C.‘Xiao Feng’and PLBs of C.‘Gong Fen Jia Ren’,their final death rates of 40 Gy irradiation were 100%,96.15%,92.00%and 95.19%,respectively.The rhizomes irradiated with 20 Gy in C.17-33 and PLBs irradiated with 40 Gy in C.‘Gong Fen Jia Ren’were recoved to grow after cultured for 160 days and 240 days,respectively.The results of SSR marker analysis showed that no polymorphic bands were amplified in the rhizomes of 0 Gy irradiation in C.17-33 and the PLBs of 0 and 40 Gyirradiation in C.‘Gong Fen Jia Ren’,while two polymorphic bands were produced in the rhizomes of 20 Gy irradiation in C.17-33,with the polymorphism rate being 2.0%.2.Using ge Norm,Norm Finder and Best Keeper software,the expression stability of Rps3,GAPDH,ACT,EF1α,Ch ACT,Ch TUB,Ch UBQ,Ch EF-1a,Chrpo B,Rps19,18S,Rpo B,TUB,26S and Psa B genes were evaluated.The results indicated that Rps19 and Ch UBQ genes could be used as internal reference genes for gene expression analysis in root,pseudobulb and leaf of C.‘Jun Hao’and its mutants.3.Using Illumina Hiseq X technology,transcriptome sequencings of leaves and mixed samples of roots and pseudobulbs of C.‘Jun Hao’and C.‘Xian Yi Jun Hao’were performed,a total of 75,985 unigenes were obtained,among which the number of differentially expressed genes（DEGs）was 13059,with 4034 upregulated genes and 9025downregulated genes.There were 8808 DEGs in leaf,among which 2415 genes were upregulated and 6393 genes were downregulated.KEGG analysis showed that DEGs in leaf enriched in 124 biological metabolic pathways,among them there were five pathways related to thread art mutation on leaf,which were anthocyanin synthesis,photosynthesis-antenna protein,porphyrin and chlorophyll metabolism,flavonoid biosynthesis and photosynthetic pathway.There were eight,seven,three,sixteen and fourteen DEGs in the above five pathways,respectively.4.Twenty DEGs were chosed from the pathways related to thread art mutation on leaf and subjected for q RT-PCR analysis.The results suggested that CYP76B10 and PSBO genes were possibly involved in thread art mutation on leaf in C.‘Jun Hao’.Through the above research,the effect of¹²C⁶⁺heavy ion radiation on the survival,differentiation and mutation of intermediate propagule in Cymbidium were preliminarily clarified,the candidate gene caused the occurrence of thread art mutation on leaf in C.‘Jun Hao’were found.These lay a foundation of further study on molecular mechanism of mutation induced by heavy ion irradiation,the establishment of effecient mutation breeding technological system by heavy ion irradiation and creation of breakthrough cultivars in Cymbidium.更多还原