Study On The Function Of COTI1 And COTI2 Of Cassia Obtusifolia In Resistance To Pests,Drought And Salt Stress

Cassia obtusifolia L was a traditional herbal medicine,which belonged to an annual legume,the main medicinal part is the dry mature seeds,which contains higher Kunitz trypsin inhibitors.With the study on the genes of Cassia obtusifolia L,we has successfully isolated the Kunitz-type gene COTI1 and COTI2 from Cassia seed.Through in vivo and in vitro experiments as well as overexpression of transgenic Arabidopsis,studied the resistance of insect,salt and drought stress of the target gene,which laid the foundation for the study of plant resistance and insect resistance.The main results were as follows:COTI1 was purified by prokaryotic expression.The purified protein COTI1 was fed on Pieris rapae to observe the growth phenotype of the larvae,found that the COTI1-fed larvae grew slower and even died compared with the normal larvae.By transcriptome data and RT-qPCR analysis found that control and experimental larvae digestive midgut and digestive-related genes were differentially expressed,the relative expression and RPKM value of control group was significantly higher than the experimental group.This result laid foundation for the research of insect resistance and insect resistant biological agents.Our lab has been cloned the sequence of Kunitz type trypsin inhibitor2 gene in C.obtusifolia(COTI2),then constructed the eukaryotic expression recombinant plasmid COTI2-pBI121 and transformed into Agrobacterium GV3101 by freeze-thawing,and expanded.Transfer COTI2 gene into Arabidopsis through inflorescence,and screened positive seedlings by kanamycin.Arabidopsis seedlings were inoculated on 1 / 2MS agar plates which contain 100 m M,150 mM NaCl and 1%,2% PEG to stress.The result showed that the COTI2-transgenic Arabidopsis grows stronger than the wild-type Arabidopsis,total root length,lateral root length and lateral root density were significantly better than wild-type Arabidopsis.According to the analysis of root scanning analyzer WinRHIZO,under the condition of drought stress,the length of lateral root of COTI2-transgenic Arabidopsis was 1.246 times as wild-type Arabidopsis.Under salt stress,the length of lateral root of COTI2-transgenic Arabidopsis was 1.577 times as wild-type Arabidopsis.Secondly,the germination rates of COTI2-transgenic seeds and wild-type seeds under2%,3% PEG stress were 53% and 24%,45% and 11%,respectively.Under 100 mM and150m M NaCl stress,the germination rate of the transgenic seeds was 84.7% and 26.7%,while wild-type was 76% and 18%.Therefore,compared with the wild-type,the seeds of COTI2-transgenic Arabidopsis have higher germination rate under drought and salt stress.COTI2-transgenic Arabidopsis and wild-type Arabidopsis were transplanted in nutrition soil,then 100 mM,150mM NaCl and 2%,3%PEG were used to simulate salt stress and drought stress,respectively.The result showed that when 100 mM NaCl stressed for 8days,the height of COTI2-transgenic Arabidopsis was 150% compared with wild-type Arabidopsis.When the stress up to 150 m M NaCl,the height of transgenic Arabidopsis was128% as wild-type Arabidopsis.Simulated drought stress by 2%PEG for 8 days,transgenic was 111% of wild-type,moreover,the plant height of transgenic Arabidopsis was 114% as wild-type when simulated drought by 3%PEG for 4 days.Thus,compared with wild-type,COTI2-transgenic Arabidopsis has stronger viability under stress,this result was useful for the study of plant resistance and good breeding.Simultaneously,the relative water content of leaves was measured.The results showed that when the 100 mM NaCl stressed for 8 days,the relative water content of COTI2-transgenic Arabidopsis was 108% as wild-type Arabidopsis.And 150 mM NaCl salt stress after 4 days,COTI2-transgenic Arabidopsis was 104% of wild-type.2% PEG simulated drought for 8 days,COTI2-transgenic Arabidopsis leaves relative water content was 109% of the wild-type Arabidopsis,and the relative water content of COTI2-transgenic Arabidopsis was 105% as wild-type under 3%PEG stress for 4 days.RT-qPCR results showed that COTI2 was differentially expressed under different stress conditions.The relative expression level of COTI2 was the highest when 1% PEG stress,and then decreased,while the expression level increased with the increase of NaCl concentration under salt stress.So,COTI2 has higher relative expression in salt and drought stress compared with no stress.Extracted the protein of COTI2-transgenic and wild-type Arabidopsis under different stress,and detected the concentration.The result showed the inhibitory activity ratio of COTI2-transgenic Arabidopsis protein was 1.37 times that of wild-type Arabidopsis when there is no stress,and the inhibitory activity ratio of COTI2-transgenic Arabidopsis proteinwas 2.54 and 1.80 times that of wild-type Arabidopsis,respectively,under drought stress and salt stress,the transgenic Arabidopsis was significantly higher than the wild type.So,COTI2 protein has an obvious inhibitory effect on the activity of trypsin and other serine proteases,plays an important role in the stress resistance of plants,etc.Transcriptome sequencing analysis of Arabidopsis roots showed that genes related to stress resistance in transgenic and wild-type Arabidopsis were significantly differently expressed under salt stress and drought stress.The expression levels of Hsp20,Hsf and R proteins in the transgenic were significantly up-regulated under drought stress,which increased the plant's ability to resist drought.At the same time,salt-tolerant proteins,glutathione S-transferases,and Kunitz trypsin inhibitors also increased significantly in salt stress,which increased the ability of Arabidopsis to resist salt,indicating that COTI2-transgenic Arabidopsis has a higher resistance to stress than wild-type.Constructed recombinant eukaryotic expression vector COTI2-pYES2,transformed into Saccharomyces cerevisiae BY4741,and identified positive clones,which laid the foundation for subsequent eukaryotic expression and protein function research.