论文部分内容阅读
目的:以高耐盐碱多年生沙漠经济灌木黑果枸杞为研究材料,对其在盐碱胁迫处理下的生理指标进行测定,确定转录组测试的时间。通过转录组分析挖掘潜在抗逆基因,并挖掘全转录组水平的分子标记。旨在为黑果枸杞的优良基因资源利用、野生品种保护和新品种培育提供理论依据和实践指导。创新点:首次对黑果枸杞进行盐碱胁迫下的生理指标变化和全转录组水平的基因表达变化进行分析,并基于转录组进行大规模简单重复序列标记开发和验证,并将所获取的分子标记应用到9个野生群体进行遗传多样性分析。方法:采用双通道PAM-100荧光仪研究盐碱胁迫对黑果枸杞P700(PS I)和叶绿素荧光(PS II)的影响;通过盐碱胁迫下丙二醛(MDA)含量、超氧化物歧化酶(SOD)和过氧化物酶(POD)活性变化选定转录组测序(RNA-seq)取样时间;采用Illumina高通量测序平台进行转录组从头测序;选取20个基因采用荧光定量聚合酶链式反应(PCR)法进行基因表达分析;基于转录组序列组装结果进行简单重复序列扫描;采用聚丙烯酰胺凝聚和毛细管电泳法鉴定引物多态性,选取其中11对多态性引物应用于遗传多样性分析。结论:通过对对照以及混合盐碱处理的黑果枸杞无菌苗进行生理和生化测试,结果选定处理6小时为取样点。RNA-seq结果共获得68 063个unigene,平均长度为877 bp,其中4096个基因在混合盐碱处理下表现为上调,4381个表现为下调。随机选取24个基因进行荧光定量表达分析,结果显示,荧光定量表达结果与RNA-seq结果呈显著正相关。基于转录组测试数据,在7940个基因中挖掘出9216个简单重复序列标记,对其中77个进行检测,显示有68个位点清晰存在,选取其中11个多态性位点对来自西北四个省份或自治区的9个野生种质资源进行遗传多样性分析,结果显示分析可靠。
OBJECTIVE: To determine the physiological index of the black walnut Chinese wolfberry, a perennial desert economic shrub with high salt-tolerance and saline-alkali resistance, and determine the time of transcriptome test. Potential stress-resistant genes were excised by transcriptome analysis and molecular markers at the level of whole transcriptome were excavated. The purpose is to provide the theoretical basis and practical guidance for the utilization of excellent gene resources of black wolfberry fruit, the protection of wild varieties and the cultivation of new varieties. Innovative point: For the first time, we analyzed the change of physiological index and the expression level of transcriptome under the condition of saline-alkali stress, and developed and validated the large-scale simple repeated sequence marker based on the transcriptome. The obtained molecule The markers were applied to 9 wild populations for genetic diversity analysis. Methods: The effects of salt-alkaline stress on PS7 and PS II of Lycium barbarum L. were studied by dual-channel PAM-100 fluorimetry. The contents of malondialdehyde (MDA), superoxide dismutase (SOD-POD) activity was selected to select the RNA-seq sampling time; sequencing was carried out using Illumina high-throughput sequencing platform for de novo sequencing; 20 genes were selected by fluorescence quantitative polymerase chain reaction (PCR) method for gene expression analysis; simple sequence repeat based on the assembly of the transcriptome sequence; primer polymorphism identification by polyacrylamide coalescence and capillary electrophoresis, and eleven pairs of polymorphic primers were selected for genetic diversity Sexual analysis. Conclusion: Physiological and biochemical tests were carried out on the control and mixed salt-alkali-treated blackberry Lycium barbarum germplasm. The results showed that 6 hours was selected as the sampling point. A total of 68 063 unigene were found with RNA-seq. The average length was 877 bp. Among them, 4096 genes were up-regulated in mixed salt-alkali treatment and 4381 were down-regulated. A total of 24 genes were randomly selected for fluorescence quantitative analysis. The results showed that there was a significant positive correlation between the results of quantitative fluorescence and RNA-seq. Based on the transcriptome test data, 9,216 simple repeat markers were excavated from 7,940 genes, of which 77 were detected, indicating that 68 loci were clearly present. Eleven polymorphic loci were selected from four Nine wild germplasm resources in provinces or autonomous regions were analyzed for genetic diversity and the results showed that the analysis was reliable.