The Functional Study Of Tomato NP24 Gene In Grey Mould And Drought Stress Resistance

Tomato is an important economic crop cultivated in a large area in the world and its fruit has high nutritional value.However,tomato rot caused by the disease is the main cause of serious economic loss.Botrytis cinerea,as one of the most harmful fungal diseases,is the main pathogen of tomato postharvest diseases.After the invasion of Botrytis cinerea,it will rapidly propagate in the plant and produce a large number of hyphae inside and on the surface of the plant,leading to dehydration and decay.Therefore,the use of genetic engineering technology to improve plant disease resistance has an important application value.Pathogenesis-related proteins（PR proteins）are secreted by plants under stress,and the presence of pathogenic molds and fungi induces the synthesis of PR proteins.The course related protein NP24 in this study belongs to the PR-5 family.Some studies have demonstrated that NP24 protein is involved in plant disease resistance in vitro through biochemical experiments and can also be induced by environmental stress.However,the function of NP24 in the anti-fungal and stress response in plants in vivo is still unclear.In this study,the role of tomato NP24 gene in Botrytis cinerea and drought resistance was studied by using tomato NP24 gene overexpression and RNAi silencing plants.The following research results are obtained:First,the expression of NP24 gene in tomato tissues was detected by qRT-PCR.The results showed that the expression level of NP24 was relatively high in fruit,leaf and root.With fruit development and ripening,the expression level of NP24gene increased gradually from damaged fruit（BR）,orange fruit（BR3）,to red ripe fruit（BR7）,and reached the highest expression level in red ripe fruit（BR7）,suggesting that NP24 gene may play a role in the ripening process of tomato fruit.Second,the response of NP24 gene to abiotic stress（drought,salt）and different hormone treatments was analyzed by qRT-PCR.The results showed that drought,salt stress,salicylic acid（SA）and ethylene（ETH）treatments could significantly induce the expression of NP24 gene,suggesting that NP24 gene may be involved in the response to biological and abiotic stress in tomato.Third,the expression of NP24 gene was analyzed by qRT-PCR at different time points after inoculation with Botrytis cinerea.The results showed that the expression of NP24 was strongly induced by Botrytis cinerea.Leaves of wild-type（WT）and NP24 overexpressed（OE）plants were inoculated with B.cinerea.Compared with WT,OE plants had smaller lesions,higher activities of defensive enzymes（CHI,Glu and PPO）,and lower contents of hydrogen peroxide（H₂O₂）and superoxide anion（O₂·^-）.The results indicated that the overexpression of NP24 gene could improve the antioxidant capacity of tomato leaves and inhibit the growth of Boerescens cinerea.Fourth,the expression of salicylic acid（SA）/jasmonic acid（JA）pathway and disease-resistance related genes in wild type and transgenic leaves inoculated with Botrytis cinerea were analyzed by qRT-PCR.The results showed that the overexpression of NP24 gene enhanced the expression of key JA biosynthesis enzymes LOXC and AOC,and up-regulated the expression of disease-resistant signaling factor Pi 1（protease inhibitor）gene,which can be induced by JA.The expression of transcription factors MYC2 and JAZ1（jasmonate-zim-domain protein 1）,which perform internal energy,was decreased,suggesting that NP24 may positively regulate Botrytis cinerea resistance through JA signaling.However,the expression levels of PR1,PAD4 and EDS1 genes in SA signaling pathway were decreased,indicating that SA pathway may play a certain antagonistic role.The expression levels of pathogen-related protein,catalase gene and endo chitinase EP3 in OE plants were significantly increased.It was speculated that NP24 protein might play a role through JA signaling pathway and enhance the basic disease resistance pathway in tomato defense against cinerea cinerea.Fifth,by inoculated wild-type and transgenic mature fruits with Botrytis cinerea,the post-harvest resistance of NP24 protein to Botrytis cinerea was analyzed.The results showed that the overexpressed fruits showed significantly enhanced resistance to Botrytis cinerea,while the RNAi fruits were very sensitive to Botrytis cinerea.Meanwhile,the accumulation of Boeris cinerea in overexpressed fruits was significantly lower than that in wild type fruits,while RNAi was now higher than that in wild type fruits.The results showed that over expression of NP24 gene could enhance the resistance of tomato fruit to cinerea cinerea.Sixth,under drought stress,NP24 overexpressed plants had better growth state and higher survival rate after rehydration than wild-type plants,while RNAi plants had the opposite effect,indicating that NP24 was involved in drought stress process at the same time.Overexpression of NP24 gene significantly improved the drought resistance of tomato seeds during germination,and reduced the water loss rate of leaves.Seventh,the injury degree of transgenic lines overexpressing NP24 was less than that of wild type under PEG stress,Compared with the wild type,the relative water content（RWC）,proline content and antioxidant enzyme activity were higher,the contents of hydrogen peroxide and malondialdehyde in plant leaves were lower,and the expression levels of several stress-related genes were significantly up-regulated.However,RNAi plants showed an opposite trend to drought stress.These results indicated that NP24 gene was a positive regulator of drought stress resistance in plants.In conclusion,the results of this study indicate that NP24 gene can improve the resistance of tomato leaves and fruits to Botrytis cinerea.And enhance the resistance of tomato plants to drought stress.NP24 is a potential gene resource for disease resistance and drought tolerance improvement of tomato.