薄壳山核桃花粉LTP基因的克隆和功能分析

Cloning and functional analysis of LTP gene in pecan pollen

【目的】比较薄壳山核桃Carya illinoinensis(Wangenh.)K.Koch和浙江山核桃Carya cathayensis Sarg花粉萌发前后蛋白质谱的差异,筛选出可能在山核桃属植物花粉萌发中起重要作用的显著差异蛋白,并分析其生物学特性和基因表达特点。【方法】基于iTRAQ技术比较两者花粉离体萌发前后蛋白质组变化的差异,筛选出显著差异蛋白,据此信息克隆和分析差异最为显著的基因,再利用已经建立的薄壳山核桃离体萌发体系和原核细胞融合蛋白表达系统来初步验证LTP(lipid transfer proteins)在花粉萌发中的功能。【结果】比较2种植物萌发前后的蛋白比值,发现有部分蛋白在2种植物中花粉萌发前后的变化规律是相反的,其中非特异脂蛋白(Non-specific lipid-transfer protein)比值达18.6倍。经过薄壳山核桃LTP同源基因的克隆和表达分析得出2个基因片段,将其命名为LTP1和LTP2,且LTP1的稳定性更好,将LTP1加入花粉萌发体系后发现LTP1对薄壳山核桃花粉萌发率和花粉管长度无显著影响。【结论】通过比较两者花粉萌发前后的蛋白质谱的差异,筛选出可能在山核桃属植物花粉萌发中起重要作用的非特异性脂质转移蛋白LTP,克隆了薄壳山核桃CiLTP1和CiLTP2基因,分析了其生物学特性和基因表达特点,但在花粉离体萌发体系中未发现体外表达的CiLTP1蛋白有影响花粉萌发的功能。研究可为揭示山核桃属植物花粉萌发规律提供重要参考。

【Objective】The protein profiles of pecan(Carya illinoinensis(Wangenh.)K.Koch)and Chinese hickory(Carya cathayensis Sarg)were compared before and after pollen germination,screening for significantly different proteins that may play a role in pollen germination.The biological properties and gene expression characteristics of these proteins were then analyzed.【Method】The iTRAQ technology was used to compare the differences of proteome changes of the two Carya plants before and after pollen in vitro germination.The significantly different proteins were identified and their genes were cloned and analyzed.Using the established pecan in vitro germination system and prokaryotic fusion protein expression system,the function of LTP in pollen germination was preliminarily verified.【Result】A comparison of protein ratios showed that some proteins had opposite patterns of change before and after pollen germination in these two plants.This included an 18.6-fold ratio for non-specific lipid-transfer protein.Two gene fragments named CiLTP1 and CiLTP2 were obtained through gene cloning and the CiLTP1 showed better stability in theory.The expression analysis in pecan indicated a high correlation between the LTP homologous genes and pollen germination.After introducing LTP1 into the pollen germination system,it was found that LTP1 does not significantly affect the pollen germination rate and pollen tube length of pecan.【Conclusion】LTP(lipid transfer proteins)were screened out by protein profiles of two species before and after pollen germination.CiLTP1 and CiLTP2 were cloned from pecan and their high correlation with pollen germination was confirmed at the gene expression level.However,the CiLTP1 protein expressed in vitro did not show any influence on the in vitro germination of pecan pollen.This study is an important reference for revealing the pollen germination law of Carya plants.