金柑MLP2-2基因抑制蛋白酶活性的功能分析

Functional analysis of MLP2-2 in kumquat:the inhibitory effect on proteinase activity

MLP2–2基因是存在于金柑中的一种H2B蛋白互作基因。对金柑MLP2–2基因进行生物信息学分析,发现MLP2–2蛋白序列中存在跨膜结构域,与Kunitz蛋白酶抑制剂具有同源性;对2年生金柑幼苗进行冷驯化,提取叶片RNA,反转录获得MLP2–2基因的cds序列,转入pBRT7质粒构建pBRT7–MLP2–2过表达载体后,侵染Rosetta(DE3)大肠杆菌工程菌,获得目的蛋白,并检测MLP2–2蛋白对金柑蛋白提取液的蛋白降解率。结果表明,MLP2–2抑制了16%的金柑蛋白质的降解,说明MLP2–2蛋白具有蛋白酶抑制剂活性。

The miraculin-like protein gene MLP2-2 is a H2B protein interaction gene in kumquat.Bioinformatics analysis of the MLP2-2 gene found that the coded protein has a transmembrane domain and has homology with the Kunitz protease inhibitor.Two-year-old kumquat seedlings were subjected to cold acclimation,and the leaf RNA was extracted to obtain the cds sequence by reverse transcription to construct the pBRT7-MLP2-2 over-expression vector,then it was transferred to Rosetta(DE3)Escherichia coli engineered bacteria to express the target MLP2-2 protein.The effect of MLP2-2 on protein degradation rate of total protein extraction from kumquat leaves was detected.The results show that MLP2-2 inhibited kumquat protein degradation by 16%,indicating MLP2-2 have protease inhibitor activity.