菠萝蜜果实PG基因的克隆与表达分析

为明确PG基因在菠萝蜜果实后熟软化过程中的作用,本研究以‘海大2号’菠萝蜜果实为材料,采用0.5 mg/L 1-MCP和1000 mg/L ETH处理,研究了室温（20℃）条件下果实成熟过程中硬度和果胶的动态变化,克隆获得4个PG基因,并对其进行了生物信息学和表达分析。结果表明：随着菠萝蜜果实的成熟,果肉硬度快速下降,可溶性果胶和离子型果胶不断增加,共价态果胶有所下降;ETH处理促进了果实WSP和ISP含量的上升,加速了软化进程,1-MCP处理抑制了贮藏前期果肉硬度的下降,推迟了软化进程,但明显提高了3种果胶的含量。AhePG1~AhePG4基因的开放阅读框（ORF）长度1221~1434 bp,编码406~477个氨基酸。AhePG1蛋白含有4个保守结构域（Ⅰ~Ⅳ）,AhePG2和AhePG3只含结构域Ⅰ和Ⅱ,AhePG4缺失结构域Ⅲ;AhePG基因分别与桃（AF095577.1）、菜豆（XM_007162208.1）、葎草（MN971583.1）、菜豆（XM_007151391.1）PG基因编码的氨基酸序列的亲缘关系较近,相似度分别达75.63%、73.11%、79.71%、70.75%。qRT-PCR分析结果显示：AhePG1基因在果实成熟前期表达量低,在后期高表达,而AhePG2/3/4基因的表达量在果实成熟过程中总体较低。ETH处理抑制了AhePG1基因的表达量,而1-MCP处理延缓了4个AhePG基因表达量的增加,但增加了成熟后期AhePG2、AhePG3、AhePG4基因的表达。相关性分析发现,果肉硬度与水溶性果胶含量和AhePG1基因表达呈显著和极显著负相关,而水溶性果胶含量又与AhePG1基因表达呈显著正相关。本研究说明,菠萝蜜果实的软化与果胶降解有关,AhePG1可能是菠萝蜜果实果胶降解和果实软化的关键PG基因之一,控制着果实成熟后期的软化;1-MCP处理能延缓菠萝蜜果实的成熟,但并不影响果实后期成熟时的软化,AhePG1、AhePG2、AhePG3、AhePG4基因可能对其软化均有作用。

Cloning and Expression Analysis of PG Gene in Jackfruit

To clarify the role of PG gene in the process of jackfruit ripening and softening, using ‘Haida 2’ jackfruit as the material, the dynamic changes of the hardness and pectin were studied at room temperature (20℃) during the ripening of the fruits treated with 0.5 mg/L 1-MCP and 1000 mg/L ETH, four polygalacturonase genes from ‘Haida 2’ fruits were cloned, and the bio-informatics and expression were analyzed. The results showed that with the ripening of the fruit, the firmness of the pulp droped rapidly, the soluble pectin and ionic pectin continued to increase, and the covalent pectin decreased. ETH treatment promoted the increase in WSP and ISP content and accelerated the process of fruit softening. 1-MCP treatment inhibited the decrease of pulp firmness in the early stage of fruit storage, delayed the process of fruit softening, but significantly increased the content of three types of pectins. The length of the open reading frames (ORF) of AhePG1~AhePG4 genes were 1221-1434bp, encoding 406-477 amino acids. AhePG1 protein contained four conserved domains (I-IV), AhePG2、AhePG3 only contained domains I and II, and AhePG4 lacked domain III. The relationship among amino acid sequences encoded by AhePG genes and PG genes from peach (AF095577.1), Phaseolus vulgarise (XM_007162208.1), Humulus (MN971583.1), and Phaseolus vulgarise (XM_007151391.1) was close, with similarities of 75.63%, 73.11%, 79.71% and 70.75%, respectively. The results of qRT-PCR analysis showed that the expression level of AhePG1 gene was low in the early stage and high in the later stage of fruit ripening. The expression level of AhePG2/3/4 genes was generally lower. ETH treatment inhibited the expression of AhePG1 gene, while 1-MCP treatment delayed the increase of four AhePGs expression, but increased the expression of AhePG2, AhePG3 and AhePG4 genes in the late stage of maturity. The result of correlation analysis showed that pulp firmness had a significant and very significant negative correlation with water-soluble pectin content and AhePG1 gene expression, and water-soluble pectin content had a significant positive correlation with AhePG1 gene expression. It showed that the softening of jackfruit fruit was related to the degradation of pectin, and AhePG1 maight be one of the key PG genes in jackfruit fruit pectin degradation and fruit softening, which controlling the softening of the fruit at the later stage of maturity.1-MCP treatment could delay the ripening of jackfruit fruit, but did not affect the softening of the fruit during the later ripening period, which meight be affected by AhePG genes.