Research on the Construction and Function of a Chlorophyll Degradation Recombinant Engineering Strain  

作　　者：Zongcan YANG Zhan ZHANG Jianbin YE Yufu PENG Xiangzhen LIU Hongxia WANG 

机构地区：[1]Technology Center, China Tobacco Henan Industrial Co., Ltd. [2]School of Food and Biological Engineering, Zhengzhou University of Light Industry

出　　处：《Agricultural Science & Technology》2017年第10期1788-1794,共7页农业科学与技术（英文版）

基　　金：Supported by the Planning Project for the Scientific Research and Technological Development of China Tobacco Henan Industrial Co.,Ltd.(ZW201435)

摘　　要：In order to effectively reduce the chlorophyll content in flue-cured tobacco, improve the overall quality of tobacco leaves, chlorophyllase gene was cloned from Arabidopsis thaliana. After the expression of the expression vector in E. coil, the recombinant engineering strain was obtained. Afterwards, IPTG （isopropy-β-D-thiogalactopyranoside）was used to induce the goal protein, and the chlorophyllase activity of the recombinant engineering strain was measured, so as to investigate its degradation effect on the chlorophyll in the extracts of tobacco leaves. The results were as follows： （1） the amplified chlorophyllase gene At- CLH1 constructed the expression vector pET28a-AtCLH1 successfully, obtaining the recombinant engineering strain; （2） induced under 30 ℃ for 22 h, the strain could well express the recombinant protein AtCLH1 with 0.5 mmol/L IPTG, and the molecular weight was about 35 kDa; （3） the strain showed good chlorophyllase producing capability, and the activity of the produced chlorophyllase could reach up to 24.9 U/mL, which could degrade the chlorophyll in tobacco extract and had a good application prospect in improving the quality of low quality tobacco; （4） based on the results of orthogonal test, the enzyme extract from the strain was added to the tobacco leaf surface, which could make the degradation rate of chlorophyll in the tobacco leaf reach 17.06% under the temperature of 37 ℃ at the humidity of 75% for 48 h; （5） after treated by the enzyme liquid, the test tobacco showed increase in the content of aromatic substances, enhancement of tobacco fragrance quality and amount, significant decrease of offensive odor and irritation, significant improvement of agreeable aftertaste, making the overall sensory quality of the tobacco leaf significantly improved.In order to effectively reduce the chlorophyll content in flue-cured tobacco, improve the overall quality of tobacco leaves, chlorophyllase gene was cloned from Arabidopsis thaliana. After the expression of the expression vector in E. coli, the recombinant engineering strain was obtained. Afterwards, IPTG(isopropy-β-D-thiogalactopyranoside)was used to induce the goal protein, and the chlorophyllase activity of the recombinant engineering strain was measured, so as to investigate its degradation effect on the chlorophyll in the extracts of tobacco leaves. The results were as follows:(1) the amplified chlorophyllase gene AtCLH1 constructed the expression vector p ET28 a-At CLH1 successfully, obtaining the recombinant engineering strain;(2) induced under 30 ℃ for 22 h, the strain could well express the recombinant protein At CLH1 with 0.5 mmol/L IPTG, and the molecular weight was about 35 k Da;(3) the strain showed good chlorophyllase producing capability, and the activity of the produced chlorophyllase could reach up to 24.9 U/m L, which could degrade the chlorophyll in tobacco extract and had a good application prospect in improving the quality of low quality tobacco;(4) based on the results of orthogonal test, the enzyme extract from the strain was added to the tobacco leaf surface, which could make the degradation rate of chlorophyll in the tobacco leaf reach17.06% under the temperature of 37 ℃ at the humidity of 75% for 48 h;(5) after treated by the enzyme liquid, the test tobacco showed increase in the content of aromatic substances, enhancement of tobacco fragrance quality and amount, significant decrease of offensive odor and irritation, significant improvement of agreeable aftertaste, making the overall sensory quality of the tobacco leaf significantly improved.

关 键 词：Tobacco leaf Degradation of chlorophyll Recombinant Engineering strain Protein expression Activity of chlorophyllase Orthogonal test Aroma substances Sensory quality 

分 类 号：Q943.2[生物学—植物学] S572[农业科学—烟草工业]