樟树钙调蛋白基因CAM的克隆与表达
Cloning and expression of CoCAM gene in Cinnamomum Camphora
当今，干旱现象频发，影响的范围极广，持续的时间很长。干旱胁迫对植物的危害很大。钙调蛋白(calmodulin，CAM)，是一种耐热的小分子蛋白质，钙调蛋白本身是没有酶活性，但是与钙离子结合后，便可作为第二信使去调节植物的各种生理活动。CAM对植物生长发育有重要作用，它能应对包括干旱在内的许多生物和非生物胁迫，在干旱胁迫下高度表达。本课题通过从樟树中克隆CAM基因，分析它与其他植物CAM基因的同源性，对该基因进行序列分析，以及研究该基因在干旱胁迫下的表达模式，为樟树的干旱生理研究及耐旱樟树新品种选育奠定基础。研究成果如下： (1)CoCAM1与CoCAM2基因序列总长都为454bp，都编码149个氨基酸。CoCAM1与华盖木、无油樟、罂粟、油棕的同源性80％以上，其中与华盖木同源性尤为显著，达到了90.60％的高水平。CoCAM2与华盖木、澳洲坚果、蒂罗花、莲的同源性80％以上，其中与华盖木同源性尤为显著，达到了91.28％的高水平。 (2)预测出CoCAM1的氨基酸分子式为C727H1144N190O255S10，分子量为16946.80，等电点4.14，不稳定系数27.39.CoCAM2的氨基酸分子式为C728H1146N190O255S10，分子量为16960.83，等电点4.15，不稳定系数29.58.预测出CoCAM1和CoCAM2均定位于细胞核，均无信号肽，均为亲水蛋白质; (3)CoCAM1蛋白质的二级结构：α螺旋占61.74％，β折叠占5.37％，β转角占9.40％和无规则卷曲占23.49％。CoCAM2蛋白质的二级结构：α螺旋占64.43％，β折叠占3.36％，β转角占8.72％和无规则卷曲结构占23.49％; (4)CoCAM1和CoCAM2蛋白质都具有哑铃型的三级结构，N端和C端有两个相似的球型结构域。并且每个球型结构域中存在两个EF-Hand结构域。 (5)荧光实时定量PCR试验显示，在干旱胁迫下，CoCAM1和CoCAM2的表达量均增加。
Nowadays,drought phenomena occur frequently,with a wide range of impacts and a long duration.Drought stress poses great harm to plants.Calmodulin(CAM)is a heat-resistant small molecule protein that does not have enzymatic activity.However,when it binds to calcium ions,it can act as a second messenger to regulate various physiological activities in plants.CAM plays an important role in plant growth and development,as it can cope with many biotic and abiotic stresses,including drought,and is highly expressed under drought stress.This project aimed to clone CAM gene from Cinnamomum Camphora,analyze its homology with other plant CAM genes,perform sequence analysis on the gene,and study its expression pattern under drought stress,laying the foundation for the study of drought physiology in Cinnamomum Camphora and the breeding of drought tolerant new varieties.The research results were as follows:(1)The total length of the CoCAM1 and CoCAM2 gene sequences was 454 bp,both encoding 149 amino acids.CoCAM1 had over 80％homology with Manglietiastrum sinicum,Amborella trichopoda,meconium,and oil palm,with a particularly significant homology of 90.60％with Manglietiastrum sinicum.CoCAM2 had a homology of over 80％with Manglietiastrum sinicum,Macadamia,Tilapia flowers,and lotus,with a particularly significant homology of 91.28％with Manglietiastrum sinicum.(2)The predicted amino acid molecular formula of CoCAM1 was C727H1144N190O255S10,with a molecular weight of 16946.80,an isoelectric point of 4.14,and an instability coefficient of 27.39.The amino acid formula of CoCAM2 was C728H1146N190O255S10,with a molecular weight of 16960.83,an isoelectric point of 4.15,and an instability coefficient of 29.58.It was predicted that CoCAM1 and CoCAM2 were both located in the nucleus,without signal peptides,and are hydrophilic proteins;(3)The secondary structure of CoCAM1 protein consisted of 61.74％alpha helices,5.37％beta folds,9.40％beta turns,and 23.49％irregular curls.The secondary structure of CoCAM2 protein consisted of 64.43％alpha helices,3.36％beta folds,8.72％beta turns,and 23.49％irregular curled structures;(4)Both CoCAM1 and CoCAM2 proteins had dumbbell shaped tertiary structures,with two similar spherical domains at the N-terminus and C-terminus.And there were two EF-Hand domains in each spherical structural domain.(5)Fluorescence real-time quantitative PCR assay showed that under drought stress,the expression levels of CoCAM1 and CoCAM2 increased.
谢荣秀;刘倩怡;王荣涛;罗雅之;杨梦婷;雷蕾;
南昌工程学院水土保持学院,江西 南昌,330000##江西省樟树繁育与开发利用工程研究中心,江西 南昌,330000;南昌工程学院水土保持学院,江西 南昌,330000##江西省科学院,江西 南昌,330000;南昌工程学院水土保持学院,江西 南昌,330000##南昌工程学院研究生院,江西 南昌,330000;南昌工程学院水土保持学院,江西 南昌,330000;南昌工程学院水土保持学院,江西 南昌,330000;南昌工程学院水土保持学院,江西 南昌,330000##江西省樟树繁育与开发利用工程研究中心,江西 南昌,330000;
Cinnamomum Camphora Drought stress CAM gene Gene cloning qPCR gene expression
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