浙江红山茶花青素和脂肪酸合成相关基因克隆与表达分析

【作者】 王仲伟；

【导师】 徐立安；

【作者基本信息】 南京林业大学 ， 林木遗传育种， 2013， 博士

【摘要】 浙江红山茶是山茶科山茶属的重要树种，树形优美、冬末春初开花，花大、色彩艳丽，是非常重要的冬春开花的观赏树种；其茶油富含不饱和脂肪酸，是高质量的食用油，被称之为东方橄榄油。本研究根据转录组测序结果，利用RACE技术克隆了浙江红山茶花青素和脂肪酸生物合成途径中的关键基因，并对它们的表达模式进行了研究。研究的主要结果如下：1、利用454测序，从浙江红山茶花芽RNA中得到了46279条EST序列，其中19397条Contigs，26882条Singlets。总共24593条序列获得了注释，17952条序列划分为56个小的类别分别归入分子功能、细胞组分、分子生物学过程中。KEGG分析发现与代谢相关的序列有3844条，涉及285个代谢通路。通过与拟南芥调控信息数据库(AGRIS)的比对，发现1740条序列与拟南芥599个转录因子具有很高的同源性，它们属于51个转录因子家族。2、根据转录组注释结果，从EST数据中检测到花青素合成通路中的10个基因，利用RACE技术，从浙江红山茶花瓣的cDNA中克隆得到了10个基因中的8个（PAL、F3H、DFR、ANS、CHI、CHS1、CHS2、CHS3、C4H和UFGT）的全长cDNA序列，其中查尔酮合酶基因（CHS）有3个成员，并通过BLAST的比对和系统发育分析，验证了它们分别属于相应基因家族中的一员。实时定量分析显示10个基因在所有组织(叶、花、种子)中都有表达，其中9个基因均在叶中的表达量最高，推测花色素合成所需的中间产物可能主要在叶中合成，然后运输到花等器官中，而CHI基因在种子中表达最高，推测CHI可能在种子中还参与了其他类黄酮物质的合成。在花发育的3个阶段(花芽、半开放、全开放)，CHS1、CHS2和CHS3的表达量在花芽中最高，PAL、CHI、F3H和UFGT在半开放的时候最高，DFR和ANS的表达随着花的发育逐渐升高，在全开放阶段达到最大。与其他物种相比，浙江红山茶花不同发育阶段这些基因的表达模式既有相似之处，也有不一致的地方，表明不同物种花青素的合成模式和调控机制可能有所不同。这些有待进一步研究。3、从EST数据中检测到脂肪酸合成通路中12个基因中的11个，其中β-酮脂酰ACP合酶基因（KAS）有3个成员，利用RACE技术，克隆得到了13个基因（BC、BCCP、ACAT、ACP、KASⅠ、KASⅡ、KASⅢ、KR、HD、ER、KCS、SAD和FAD2）的cDNA全长序列。生物信息学分析证实了它们的正确性。通过qRT-PCR分析表明，除FAD2在第二阶段表达最高，随后一直维持低表达外，其他基因几乎都是在种子发育的中期（第三至第五阶段）表达最高，其中半数基因的表达在第五阶段达到最大，只有ACP、KASⅡ和KASⅢ在第三阶段比第五阶段表达水平略高，KASⅠ和SAD到第四阶段表达较高。利用气相色谱技术，对浙江红山茶种子不同发育阶段的主要脂肪酸的含量进行了检测。种子发育过程中的脂肪酸相对含量变化很大，在发育初期，亚油酸和亚麻酸的总含量明显高于油酸，随后油酸所占比例逐渐增加，从第三阶段后就成为最主要脂肪酸成分，到第七阶段时油酸含量高达75%以上，而亚油酸和亚麻酸的总含量下降到了12.5%左右。而饱和脂肪酸（棕榈酸和硬脂酸）相对含量随着种子发育逐步下降，从第二阶段的45%左右至第七阶段时已降至12%以下。SAD和FAD2为脂肪酸去饱和基因，其中SAD与油酸形成有关，FAD2与亚油酸或亚麻酸合成有关。种子不同发育阶段的油酸、亚油酸和亚麻酸变化趋势与SAD和FAD2表达量变化基本相对应，只是相应不饱和脂肪酸的含量的变化有一个滞后过程。尽管饱和脂肪酸合成相关基因基本在第三至第五阶段表达最高，而饱和脂肪酸的相对含量却在逐步下降，这是由于产生的饱和脂肪酸被SAD和FAD2转化成了不饱和脂肪酸。更多还原

【Abstract】 Camellia chekiangoleosa is an important species of genus Camellia belonging to thefamily Theaceae. The economic value of C.chekiangoleosa is largely due to its beautifulornamental flower. The flower is red which generally blooms between February and March. Inaddition, C.chekiangoleosa is used to produce high-quality edible oil from its seeds. Its oil isrich in unsaturated fatty acid. The economic value of its oil is the same as oil palm, olive,coconut, and is commonly known as “eastern olive oil”. In this research, the key enzyme genesof anthocyanins and fatty acid biosynthetic pathway were cloned, and their expression patternswere investigated. The main results were showed as following:1、454GS FLX Titanium platform was used to generate an EST dataset. About46,279sequences were obtained, and24,593were annotated. Fifty-six functional groups including17,952sequences were classified according to their molecular function, biological process, andcellular component ontologies. A total of3844unigenes assigned to285metabolic pathwaywere identified against the KEGG database. Using Blast search against the AGRIS,1,740unigenes were found homologous to599Arabidopsis transcription factor genes.2、Based on the transcriptome dataset, ten anthocyanin biosynthesis pathway genes (PAL,C4H, CHS1, CHS2, CHS3, CHI, F3H, DFR, ANS, and UFGT) were identified and cloned. Themultiple alignment and phylogenetic analysis demonstrated that each gene was a member ofcorresponding gene family. Relatively high expression levels of all the genes were observed inthe leaf except CHI, whose expression was abundant in the seeds. Hence, it was hypothesizedthat the anthocyanins were mainly synthesized in the leaves and transport to the flowers andseeds. The temporal expression analysis showed that CHS1, CHS2, and CHS3were highlyexpressed in the bud stage; F3H, CHI, PAL, and UFGT were maximally expressed in thesemi-open stage; and the expression of DFR and ANS gradually increased with flowerdevelopment. There are many similar or different patterns in various species, This could beattributed to some important differences between the types of anthocyanins produced by eachspecies and their regulatory mechanism.3、Thirteen complete-length cDNAs encoding BC, BCCP, ACAT, ACP, KASⅠ,KASⅡ,KASⅢ, KR, HD, ER, KCS, SAD and FAD2were isolated from seed by RACE. The sequenceanalysis validate their correctness. Almost all genes were highly expressed in the middle ofseed development (Ⅲ toⅤ) except FAD2. FAD2was maximally expressed in the second stage,its expression level in other stages is very low.Study of fatty acid accumulation pattern in seed development by gas chromatographyindicated that contents of fatty acids changed a lot. In the early stages, the total content oflinoleic acid and linolenic acid was higher than oleic acid, but they declined withthe subsequent development of the seed and the proportion of oleic acid increased gradually.The content of palmitic acid and stearic acid were gradual declined in seed development. SADand FAD2involved in the the formation of unsaturated fatty acids, wherein SAD catalyzed oleic acid formation and FAD2relating to the synthesis of linoleic acid or linolenic acid. Theirexpression quantities are roughly identical to the change trend of oleic acid linoleic acid andlinolenic acid. Although genes were highly expressed in the third to fifth stage, the relativecontent of saturated fatty acids was decreased gradually. This is due to SAD and FAD2convertsaturated fatty acids to unsaturated fatty acids.更多还原