Selection,Cloning And Functional Analysis Of Rice NsLTPs Genes Involved In Brown Planthopper Defense

Plant non-specific lipid transfer proteins(ns LTPs)are a class of small proteins that are widely found in terrestrial plants and can transfer lipids with broad specificity across membranes.They perform various physiological functions in different tissues and developmental processes of plants.Physiological processes such as wax synthesis and transportation,lipid secretion and signal transductions involved by ns LTPs are closely related to plant resistance to various environmental stresses,and a large number of studies have shown that ns LTPs play critical roles in pathogens and abiotic stresses such as low temperature and drought.However,there are few reports on the role of ns LTPs in plant resistance to insect pests.Thus,in this thesis we screened and cloned ns LTPs genes involved in BPH(Nilaparvata lugens)defense in rice(Oryza sativa),a monocotyledonous model plant by transcripome analysis and clarified the expression patterns of these genes,then knockout mutants were constructed by CRISPR/Cas9 system to preliminarily explore the physiological function of LTP14 in BPH defense.1 Transcriptome analysis of rice with different insect resistance infested by BPHThrough the transcriptome analysis of stem tissue of two rice varieties with different insect resistance after BPH infestation,43834 transcripts with an average length of 2047 nt were assembled.Further,by differential expression and enrichment analysis we found that the proportion of differentially expressed genes responsed to stresses in ASD7 was higher than that in TN1,and the number of up-regulated genes responsed to stresses in ASD7 was greater than that of down-regulated genes.In addition,by analyzing genes enriched in defense-related GO term whether common or specific to two rice cultivars,we found that ns LTPs genes were significantly differentially expressed between two rice cultivars after BPH infestation.Therefore,non-specific lipid transfer proteins may be involved in BPH defense in rice.2 Analysis of the expression pattern of rice ns LTPLs genesAmong the ns LTPs genes that may be involved in BPH defense based on transcriptome analysis,six of them were cloned and their spatiotemporal expression patterns were detected by RT-q PCR.These ns LTPs genes had different expression characteristics.During the reproductive growth of rice,the expression level of LTPL8 in aboveground tissues was significantly higher than that of other ns LTPs,followed by the expression level of LTPL14.The expression level of LTPL152 in root was significantly higher than that of other ns LTPs.However,during vegetative growth period,the expression level of LTPL14 in shoot was significantly higher than that of other ns LTPs genes,and the expression level of LTPL14 and LTPL152 in the shoot and leaf of the TN1 were significantly higher than that of ASD7 during the whole investigation period.3 LTPL14 mutant rice plants were constructed by CRISPR/Cas9 gene editing systemLTPL14 mutant rice plants were constructed by CRISPR/Cas9 gene editing system.A total of three homozygous mutant rice plants were indentified.The three homozygous mutants were all identified with good editing effects based on the LTPL14 gene expression levels in BPH infested or not mutants and wild type(WT)rice plants.The mutants showed no significant difference in root length and tillers compared with TN1 rice plants,but showed obviously dwarfing.In addition,the inhibited expression of LTPL14 gene in rice did not affect olfactory selection of BPH,but the number of eggs laid by BPH on mutant rice increased.The egg production of lt PL14-12 homozygous mutant was significantly higher than that of wild-type rice.