Functional Analysis Of OsbHLH35 Transcription Factor Involved In Resistance To Brown Planthopper In Rice

Oryza sativa is one of the most important food crops in our country and plays a very important role in food security.Nilaparvata lugens(brown rice planthopper,BPH)is an important pest on rice production in our country and seriously affects the yield and quality of rice.Cultivating insect-resistant variety is considered to be the best way to control BPH.But the high degree of plasticity of BPH-specific hazard-causing ability makes traditional resistance breeding and application unable to continuously control this pest.Therefore,the key to the prevention and control of BPH is to continuously discover and utilize new effective anti-BPH genes for the breeding of resistant new varieties to achieve long-lasting resistance to BPH.When the plant is attacked by herbivorous insects,it can induce an insect resistance response,and its molecular mechanism has been a research hotspot.The level of transcription of a large number of genes in plants induced by herbivore insect infestation changes,and transcription factors play an important role in plants.The bHLH transcription factor family is one of the largest families of plant transcription factors,and it is widely involved in the growth and development of plants as well as in response to biological and abiotic stresses.However,its regulation mechanism in response to rice BPH harm is not clear.In our previous work,the rice transcription factors were respectively constructed into a transcriptional activator and a transcriptional repressor by the GatewayTM large-scale cloning technology.After genetic transformation,57,751 independent transgenic rice lines respectively with functional gain or loss of the transcription factor were obtained.Based on the previous screening of insect resistance through the indoor seedling group screening method,we found that the transgenic rice material V1188 activated by the expression of the OsbHLH35 transcription factor exhibited high levels of susceptibility to BPH.What's more,the resistance of the expression inhibitor E1188 to BPH was not significantly different from that of the wild-type rice KTK.It was preliminarily deduced that V1188 was a susceptible material to BPH.In this paper,OsbHLH35 transcription factor was taken as the research object,and the transgenic rice V1188 activated by OsbHLH35 expression was used as the test material.The effect and regulation mechanism of OsbHLH35 transcription factor in the induction of rice anti-BPH was preliminarily studied.The main results were as follows:1.Based on the results of the previous laboratory screening,we used normal PCR technology to verify that V1188 and E1188 were the correct transgenic materials.Hygromycin screening confirmed that the OsbHLH35 transcription factor expression activating materials V11886,V11889,and V118810 are transgenic homozygous lines activated by OsbHLH35 transcription factor expression and can be used in the next experiments.Phenotypic observations revealed that V11886,V11889,and V118810 had no significant differences in seed germination and plant height compared to wild-type rice Kitaake(KTK).However,the root lengths of the three transgenic lines were reduced by about 42%compared with KTK,indicating that the OsbHLH35 transcription factor inhibits the rice root length after activation in rice.Whether the gene affects rice resistance by regulating rice root length remains to be further studied.2.The expression level of OsbHLH35 transcription factor in different tissues and at different stages of rice was analyzed by using real-time quantitative PCR technology.The results showed that the expression level of OsbHLH35 gene was highest in leaf sheaths at four-leaf stage and tillering stage in KTK.Its expression level in leaf sheaths of V11886,V11889 and V118810 was about 13 times of the KTK.In addition,the OsbHLH35 gene was highly expressed in V11886,V11889,and V118810 after BPH feeding,which was 12.6,13.5,and 18.7 times of the control KTK,respectively.Since BPH damages rice through leaf sheath feeding,the high expression of the OsbHLH35 transcription factor in leaf sheaths suggests that it may play a role in BPH feeding process,thus affecting rice resistance to BPH.3.Through the insect resistance biological assay,the results showed that the area of honeydew secreted by BPH on lines V11886,V11889,and V118810 was approximately 2 times compared to KTK.The survival rate of BPH on the V11886,V11889 and V118810 lines was about 80%on the seventh day and only 67%on the KTK.At the same time,the growth rate of BPH population on V11886,V11889 and V118810 strains is approximately 1.4 times compared to KTK.This indicates that BPH prefers feeding and activity on the three lines of the OsbHLH35 transcription factor expression-activated materials,suggesting that the OsbHLH35 transcription factor may inhibit the circumvention defense mechanism of rice and reversely regulate rice resistance to BPH.4.After BPH feeding,compared to KTK,the expression level of two key synthetic genes AOS2 and LOX in jasmonic acid(JA)pathway decreased significantly by 83%,74%,64%and 76%,64%,55%,respectively.In addition,the enzymatic activities of the plant defense enzymes polyphenol oxidase(PPO)and phenylalanine ammonia lyase(PAL)in V11886,V11889 and V118810 also dropped significantly by 50%,44%,48%and 35%,45%,48%compared to KTK after BPH feeding,respectively.The above results indicate that the OsbHLH35 transcription factor may inhibit the JA signaling pathway by inhibiting the induction of rice AOS2 and LOX gene expression,and at the same time inhibit the activities of major defense enzymes PPO and PAL in rice,thereby impairing rice's resistance to insects.This suggests that the OsbHLH35 transcription factor may be a negative regulator of rice resistance to BPH.This research will provide a theoretical basis for the resistance breeding of rice to BPH and the prevention and control of BPH.