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钴(Co)胁迫会对植物产生多种毒害作用。为研究其植物毒性作用机理,需构建Co胁迫条件下紫花苜蓿的实时荧光定量PCR反应体系。研究选取了6个持家基因(actin2,GAPDH,UBI,18S,MSC27,EF1-α)作为备选内参基因,以紫花苜蓿植株为实验材料,检测Co胁迫条件下备选内参基因的表达稳定性。通过RefFinder和geNorm软件分析,确定Co胁迫下紫花苜蓿最佳内参基因组合为actin2和18S。将四种根际促生菌分别接种紫花苜蓿,并使用100 mg/L CoCl_2对接种后植株进行处理,使用筛选内参引物检测生育酚合成关键基因尿黑酸叶绿醇转移酶(HPT)的转录水平。研究结果显示Co胁迫可使HPT基因转录水平发生显著下调,根际促生菌铜绿假单胞菌Pseudomonas aeruginosa 1401可降低Co胁迫对苜蓿植株的毒害作用,接种该菌株植株的HPT基因相对表达水平为对照样本的6.63倍,植株株高和生物量分别达到对照样本的140.86%和193.62%。根据测试样本中HPT基因转录水平与植株Co耐受能力的变化趋势,推测紫花苜蓿的Co耐受能力增强与生育酚的合成途径调控或存在紧密联系。
Cobalt (Co) stress will have a variety of plant toxic effects. In order to study its mechanism of phytotoxicity, real-time fluorescence quantitative PCR reaction system of alfalfa under Co stress conditions needs to be constructed. Six housekeeping genes (actin2, GAPDH, UBI, 18S, MSC27, EF1-α) were selected as candidate reference genes. The alfalfa plants were used as experimental materials to test the stability of the candidate genes under Co stress. RefFinder and geNorm software analysis, to determine the Co under stress alfalfa best internal reference gene combination actin2 and 18S. Four rhizobacrophi were inoculated with alfalfa respectively and the inoculated plants were treated with 100 mg / L CoCl 2. The transcription of uric acid transferase (HPT), a key gene for tocopherol synthesis, Level. The results showed that co-stress could significantly down-regulate the transcription level of HPT gene. Pseudomonas aeruginosa 1401 could reduce the toxicity of Co stress to alfalfa plants. The relative expression level of HPT gene was 6.63 times that of the control sample, plant height and biomass reached 140.86% and 193.62% of the control sample respectively. According to the trend of HPT gene transcription level and plant tolerance to Co in the test samples, it is speculated that the tolerance of Co to alfalfa is closely related to the regulation of tocopherol synthesis pathway.