葡萄果实着色前后代谢合成关键酶的基因表达分析

葡萄果实着色前后ABA代谢合成关键酶的基因表达分析

THE ANALYSIS OF GRAPES METABOLIC AYNTHESIS OF ABA KEY ENZYMES GENE EXPRESSION BEFORE AND AFTER THE FRUIT COLOR

作者：王延书　　导师：高东升　沈元月

山东农业大学　　果树学2011届博士

摘　要

本实验试材为玫瑰香葡萄（Vitis vinifera L.cv.Muscat Hamburg），由北京农学院葡萄种质资源圃提供。玫瑰香葡萄，又名麝香葡萄（Muscat），欧亚种，于1871年由美国传教士倪氏首先引入山东烟台，1892年又从西欧引入，是我国分布最广的品种之一，各主要葡萄产地均有栽培。此品种属中晚熟品种，植株树势生长中等，二次结果率高，丰产性较好，颗粒小，口感微甜，有玫瑰香味，肉质坚实，易运输，耐贮藏，是鲜食和酿酒兼用品种。

实验于2008年到2010年进行，共分为两部分。第一部分，花后2周开始采集果实，每7天一次，直至果实完熟，研究了玫瑰香葡萄果实的生长变化动态、相关的果实品质指标及ABA含量变化；同时探讨了9-顺环氧类胡萝卜素双加氧酶（NCED）、细胞色素P450双加氧酶（CYP）、葡萄糖基转移酶（GT）及β-葡萄糖酯酶（BG）在花后7～11周葡萄果实中表达量的变化规律。第二部分，果实着色初期进行不同浓度脱落酸（ABA）涂抹处理，处理浓度为100 mg/kg和250 mg/kg；以喷水为对照，处理7天后采样，研究了不同浓度ABA处理对葡萄果实着色及品质的影响。结果如下：

1.　涂抹ABA有利于提高葡萄果实的着色指数，随着涂抹浓度的增加，着色指数增大；葡萄成熟时的测定结果表明，ABA提高了果粒的平均单粒重，增加了果实含糖量，以250 mg/kg ABA的处理效果最好，100 mg/kg ABA次之。

2.　玫瑰香葡萄果实纵横径变化趋势基本一致，分快—慢—快三个时期。液相色谱测定表明，果实内可溶性糖主要为葡萄糖、果糖和蔗糖，葡萄糖和果糖变化趋势基本一致，自着色期初期即开始快速上升，直至完熟期，其中果糖增幅大于葡萄糖；蔗糖含量较少，呈平缓上升趋势，但增幅较小；总酸含量和pH值呈相反变化趋势，总酸含量快速下降的同时pH值迅速升高；ABA含量伴随着色期的发生而迅速增加，着色期后期达到高峰值，之后下降。说明果糖对果实成熟时甜度的提高起着重要作用，ABA与果实着色期密切相关。

3.　提取葡萄果实的总RNA，反转录为cDNA后，分别设计NCED、CYP、GT、BG四个基因的兼并引物，然后以葡萄cDNA为模板分别扩增出所需基因片段，根据克隆到的基因片段设计荧光引物，测定上述四个基因在葡萄花后7～11周表达量的变化。VvNCED1基因表达量在花后7～8周较高，第9周迅速降至最低值，10～11周又略有升高，但其值远低于初始值；随着果实着色期的启动，VvBG1基因表达量有两次快速升高的过程：第一次升高发生在花后第7～8周，第二次升高发生在花后第9～10周，且第二次上升幅度大于第一次；VvGT1在第8周迅速升至峰值，而后大幅下降，之后变化较为平稳；VvCYP1基因表达量随果实成熟度的加深而逐渐升高，第9周达到最大值，然后迅速降至极低值。从以上结果可知，玫瑰香葡萄果实着色期前期ABA的积累主要归因于VvNCED1基因的高水平表达，而后期ABA的积累则主要归因于VvBG1基因；花后第8～9周，VvGT1和VvCYP1均处于较高的表达水平，与此时ABA含量较低相吻合。

4.　对VvBG1基因进行生物信息学分析显示，此基因编码的蛋白质分子量约为57KD，PI值为6.24，其二级结构为桶状。

关键词　葡萄果实　ABA　代谢合成　基因表达

Abstract

Muscat grape (Vitis vinifera　L.cv.Muscat Hamburg) provided by Beijing　Agricultural collage’s grape germplasm nursery was studied in this research. Muscat grape belonging to Eurasia and was led into Yantai Shandong Province by American missionary-Ni shi in 1871 for the first time,and in 1892 for the second time. Muscat grape is one of the most widely distributed species in our country and nearly every grape origin had cultivation of it. This breed has the features as following displayed: middle-late in maturity,moderate in tree vigor,high quality in secondary outcome,high yield quality,fruit is small,taste sweet and take on a rose fragrance,flesh is firm and easy to transport or long-storage. Above all,this is a cultivar can be used both for eat and wine-making. The experiment was carried on between the year 2008 and 2010 which contained two parts. The first part mainly researched the　dynamics of fruit growth,fruit quality index and ABA content using the fruit collected every 7 days after 2 weeks of flowering. And also the different expression of 9-cis expoxy carotenoid dioxygenase (NCED),cytochrome P450-dioxygenase (CYP),glycosyltransferase (GT) and　β-glucose esterase using fruit collected between 7 and 11 weeks after flowering. The second part mainly studied effects of different concentrations (100 mg/kg and 250 mg/kg,the field-grown as control) of ABA treatment on fruit coloring and quality using fruit treated 7 days. The results were as follows:

1.　With smear of ABA　can be　beneficial to improve color index of grape berry. with the increasing of smear concentration,color index were improved. determination results of grape berries suggest: ABA improved average single particle weight of grape berry,increased the sugar content. Treatment effect was the best with the concentration of 250 mg/kg　of ABA,and 100 mg/kg of ABA took the second place.

2.　The trend of vertical and horizontal diameter of grape berry was almost the same,which was divided into fast-slow-fast three periods. The soluble sugars are mainly glucose,fructose and sucrose. glucose and fructose have the same changing trend which increases from the early veraison to full maturity. fructose　has a more increase amplitude than glucose. Sucrose content is less,which has a gentle increasing trend and the increase amplitude is small.　At and after veraison，pH value increases rapidly,having matching and opposite changing trend with total acid content. Similarly,ABA content increases rapidly coupling with veraison,and having a peak at two weeks after veraison,then declining until complete ripening. we can conclude that fructose plays an important part on the sweet increasing during fruit ripening. ABA is closely related with the veraison time of fruit.

3.　The RNA was extracted from the grape　berry,then inversed into cDNA. We designed degenerate primers of NCED、CYP、GT、BG,and amplified 4 gene fragments and sequencing. Based on gene fragments of four genes,we designed fluorescence primer to determinate expression changes of four genes 7 to 11weeks after flowering. The results were as follows.（1） the expression level of VvNCED1 gene was very high during 7～8 weeks after flowering,followed by a rapid decline to its lowest point,and then increased a little.（2）the expression level of VvBG1 had two quick increasing. with the onset of fruit ripening,the first increasing began 7-8 weeks after flowering. The second increasing began 9-10weeks after flowering,which has a quick increasing. (3)the expression level of VvGT1 increased quickly to the maximum at 8 weeks　after flowering,then declined quickly. the expression level of VvGT1 9-11weeks after flowering was higher than that of 7 weeks after flowering. (4) the expression level of VvCYP1 increased to its maximum from 7 to 9 weeks after flowering ,then declined to its minimum in the following two weeks. the expression level of VvCYP1 10-11weeks after flowering was less than that of 7 week after flowering. Given that the ABA continual rising during　the onset of grape berry ripening,it is considered that，before the onset of berry ripening. the ABA accumulation is attributed to high mRNA expression level of　VvNCED1;after the onset of　berry ripening,ABA accumulation is attributed to high mRNA expression level of　VvBG1. both VvGT1and VvCYP1　are in higher expression level 8-9weeks　after flowering,which is in accordance with lower content of ABA.

4.　Bioinformatics analysis was performed　to VvBG1 gene. The molecular weight was 57KD,the pI were predicted as 6.24. Confirmation prediction was like a barrel.

Key words　Grape berry　ABA　Metabolism and biosynthesis　The expression level