摘要
【目的】蜡粉是植物抵御外界胁迫的第一层保护性屏障,对西瓜果实表皮蜡粉的结构、化学成分、遗传规律进行研究,并预测控制该性状的基因,以便全面了解性状的生理生化作用,发掘候选基因。【方法】试验选用无蜡粉的西瓜自交系‘美佳选黑’(P_1)和有蜡粉的西瓜自交系‘FH’(P_2)为双亲配制杂交组合,构建六世代群体(P_1、P_2、F_1、F_2、BC_1P_1、BC_1P_2),利用扫描电子显微镜(SEM)对双亲成熟期果实表皮结构进行观察,通过气相色谱-质谱联用仪(GC-MS)对蜡粉的化学成分进行测定,以峰面积为指标,定量计算蜡粉化学成分的含量,采用BSA-seq对西瓜表皮蜡粉进行基因初定位,应用BLAST软件将定位区间内的编码基因与多个数据库比对完成基因信息注释,并通过详细的基因注释信息及突变位点分析,快速筛选候选基因。【结果】西瓜果实表皮蜡粉为灰白色,呈致密的板状结构,长度约为5μm;无蜡粉材料表皮较为光滑,无蜡粉层附着。GC-MS分析显示,蜡粉中共检测到24种脂肪族化学成分,分别属于烃类、醇类、酯类、酸类、酚类和醛类。包括烃类物质10种,含量占表皮蜡粉有效化学提取物总含量的77.72%,链长变化范围为C17—36,主要为C27、C28、C29、C32、C33、C34、C36的饱和正链烷烃;醇类物质5种,占12.60%;酯类物质5种,占0.43%;酸类物质2种,占0.53%;酚类1种,占0.80%;醛类1种,占3.99%;含量最高的5种化学成分依次为:正三十四烷、正二十九烷、1,30-三十烷二醇、正三十三烷、正二十八烷。在后代群体中,F_1、BC_1P_2群体西瓜果实表皮全部有蜡粉,F_2群体有蜡粉和无蜡粉西瓜果实的分离比符合3﹕1的孟德尔分离比例,BC_1P_1回交群体有蜡粉和无蜡粉分离比符合1﹕1的理论比,说明蜡粉的有无符合单基因显性遗传模式,有蜡粉对无蜡粉为显性。对BSA-seq数据进行SNP和In Del关联分析,关联结果取交集得到1号染色体3.16—4.84 Mb的候选区域,该区域共含有144个基因。数据库比对结果显示,在关联区域中,共138个基因有功能注释,包括10个非同义突变,1个移码突变。结合已有文献报道,其中5个非同义突变基因可能与西瓜表皮蜡粉的生成有关:Cla002367为烯酰ACP-还原酶(ECR)类基因,该类基因是特长链脂肪酸合成所必须;Cla011514、Cla002337和Cla002342为细胞色素P450(CYP)家族基因,该家族部分编码蛋白能够通过烃基羟化等反应催化脂肪族化合物的生成;Cla002353的基因注释信息为ABC转运体,ABC转运体与蜡质分子的转运息息相关。【结论】西瓜果实表皮蜡粉为板状结构,主要由特长链脂肪酸衍生成的脂肪族化合物组成。该性状是单基因遗传,有蜡粉为显性性状。BSA关联分析得到1号染色体1.68 Mb的关联区域,并预测关联区域中Cla002367、Cla011514、Cla002337、Cla002342、Cla002353这5个非同义突变基因为调控西瓜果实表皮蜡粉性状的候选基因。
[Objective]Wax powder is the first protective barrier for plants to resist external stress. In order to find out the physiological and biochemical mechanism of this trait and to get candidate genes, the structure, chemical compositions and inheritance of wax powder on watermelon fruit epidermis were investigated in this research, and the candidate genes were predicted.[Method]Six-generation populations (P1, P2, F1, F2, BC1P1, and BC1P2) were constructed by crossing the inbred line ‘Meijiaxuanhei’(in glossy) and ‘FH’(in waxy). Scanning electron microscopy (SEM) was used to observe the structure of wax powder on mature watermelon fruit epidermis. The composition of wax powder was determined by gas chromatography-mass spectrometry (GC-MS), and the contents of different chemical components were quantitatively calculated based on the peak area. The wax powder trait was preliminary mapped by BSA-seq, and BLAST software was used to annotate the coding genes in the localization interval in many databases. By analysis of detailed gene annotation information and mutation site, candidate genes were quickly screened.[Result]The color of wax powder on ‘Meijiaxuanhei’ fruit was gray-white, and the structure was compact plate shaped with about 5 μm in length, while ‘FH’ fruit was found smooth epidermis and no similar structure. 24 kinds of aliphatic compounds were detected in the wax powder, which were classified as hydrocarbons, alcohols, esters, acids, phenols and aldehydes. There were 10 kinds of hydrocarbons were found, which accounted for 77.72% of the total effective chemical extracts of epidermal wax powder and chain length ranged from C17 to C36. Among them, most of the saturated n-alkanes were C27, C28, C29, C32, C33, C34 and C36, 5 kinds of alcohols accounted for 12.60%, 5 kinds of esters accounted for 0.43%, 2 kinds of acids accounted for 0.53%, 1 kind of phenol accounted for 0.80%, and 1 kind of aldehydes accounted for 3.99%, respectively. The top five chemical components in watermelon wax powder were n-34, n-29, 1,30-triacontanediol, n-33 and n-octacosane. The fruit of F1 and BC1P2 progenies had wax, the segregation of waxy and glossy in F2 progenies corresponded to the Mendelian ratio of 3:1, and the segregation ratio in BC1P1 progenies was 1:1, so it could be concluded that the hereditary mode of waxy was the dominant heredity of single gene, and the waxy type was the dominant character. By SNP and InDel correlation analysis, 3.16-4.84Mb association region of chromosome 1 was obtained, which contained 144 genes, 138 genes in them were functionally annotated, including 10 non-synonymous mutations and 1 frameshift mutation. Combining with the literature reports, there were five non-synonymous mutation genes might be related to the formation of watermelon epidermal wax powder: Cla002367 was an enoyl ACP-reductase (ECR) gene, which was essential for the synthesis of extra-long chain fatty acids. Cla011514, Cla002337 and Cla002342 were cytochrome P450 (CYP) family genes, and some proteins encoded by the CYP family genes could catalyze the formation of aliphatic compounds through alkyl hydroxylation and other reactions;Cla002353 gene annotation information was ABC transporter, and ABC transporters were closely related to the transport of waxy molecules.[Conclusion]Wax powder on watermelon fruit epidermis was a plate structure, which was mainly composed of aliphatic compounds derived from long chain fatty acids. The trait conformed to the single gene inheritance model, and wax powder was dominant. The association region of chromosome 1 in the range of 1.68Mb was obtained by BSA correlation analysis. 5 non-synonymous mutation genes Cla002367, Cla011514, Cla002337, Cla002342 and Cla002353 were predicted as candidate genes for wax powder on watermelon fruit epidermis.
作者
龚成胜
赵胜杰
路绪强
何楠
朱红菊
豆峻岭
袁平丽
李兵兵
刘文革
GONG ChengSheng;ZHAO ShengJie;LU XuQiang;HE Nan;ZHU HongJu;DOU JunLing;YUAN PingLi;LI BingBing;LIU WenGe(Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences/National Cucurbits and Fruits Improvement Center, Zhengzhou 450009)
出处
《中国农业科学》
CAS
CSCD
北大核心
2019年第9期1587-1600,共14页
Scientia Agricultura Sinica
基金
国家重点研发计划(2018YFD0100704)
中国农业科学院科技创新工程专项经费项目(CAAS-ASTIP-2016-ZPRI)
现代农业产业技术体系建设专项(CARS-25-03)
国家自然科学基金(31471893
31672178)
关键词
西瓜
蜡粉
表皮结构
化学成分
遗传分析
基因定位
watermelon
wax powder
epidermis structure
chemical compositions
genetic analysis
gene mapping
