| Many crops are facing the threat of insect pests,and the total amount of plants lost due to the threat of pests is large in China.It is reported that the annual damage caused by pests in China’s crops is as high as nearly 50 billion catties.Tobacco(Nicotiana tabacum)is an important model plant and is widely used to study the interaction between plants and insects.Tobacco used as a model plant to study the insect resistance mechanism of plants has important theoretical and practical significance for the cultivation of insect-resistant crop varieties.Nowadays,the control of crop pests in China is still mainly based on chemical control and supplemented by biological control.But these anti-pest measures,especially chemical control,not only bring serious pollution to our environment,but also make the pests have stronger resistance.Although biological control will not cause environmental pollution,on the one hand,the synthesis of sex pheromones used to control pests is usually very difficult,and on the other hand,each pest have its own sex pheromone also restricted the application.Cultivating insect-resistant plant varieties through genetic engineering is an ideal measure for controlling pests that has emerged along with the advancement of molecular biology methods.In recent years,the first insect-resistant transgenic plants which expressed Bt toxic protein,have appeared.Although the effect of controlling pests is good,the tolerance of pests to Bt has increased year by year.That exploring the negative regulatory factors related to plant insect resistance,and then inactivating or reducing its expression through genetic engineering was considered to be promising direction for molecular breeding of insect-resistance crops in the future.In this study,cultivated tobacco was used as a research model,and the candidate negative regulatorsCytochrome P450(NtCYP450)and Jasmonoyl-L-isoleucine hydrolase 1(NtJIH1)were identified and knocked out by CRISPR/Cas9 gene editing to obtain mutants.The mutants were expanded and their insect resistance,related physiological and biochemical indexes were tested.This research mainly achieved the following research results:1.Identification and expression pattern analysis of Nt JIH1 and NtCYP450Through homologous sequence alignment,a total of 15 JIH1 genes(Nt JIH1-1-NtJIH1-15)and three candidate CYP450 genes(Nt CYP450-1,NtCYP450-2and NtCYP450-3)with insect resistance were identified in the tobacco HHDJY variety.The results of the evolutionary tree show that NtJIH1 family can be divided into 8branches;the results of multiple sequence alignments show that it mainly contains C(cysteine Acid),E(glutamic acid),H(histidine)and other active sites,moreover,the two genes NtJIH1-1 and NtJIH1-2 are the closest to the gene of NaJIH1,which has been reported in the diploid tobacco;results of gene structure show that most NtJIH1 genes contain 5 exons,and always appeared as paired genes.For NtCYP450 gene,three genes identified in this study were closest to the reported insecticidal negative regulation CYP450 gene(accession number: AF166332),which were named as NtCYP450-1,NtCYP450-2,and NtCYP450-3,respectively,which are located in the 14 th clades of the CYP45071 family;the prediction results of its active region show that the P450 domain region is retained,implying that it is highly conservative in evolution;the expression data based on microarray shows that most NtCYP450 genes have similar tissue/organ expression patterns.The expression profile of tobacco infested by the cotton bollworm showed that multiple NtJIH1 genes,including NtJIH1-1/2,-4,-5,-7,-9/10,-11,-12 and-13,were significantly up-regulated at 6h;similarly,NtCYP450-1,NtCYP450-2 and NtCYP450-3 could be also significantly up-regulated by insect chewing.The expression profile of tobacco infested by Myzus persicae showed that many genes,including NtJIH1-1/2,-3,-4,-5,-6,-7,-9/10,-12 and-15 in NtJIH1 gene family and Nt CYP450-1,NtCYP450-2 and NtCYP450-3,showed an significantly upregulation at 6h after aphid infestation.These results preliminarily suggested that some genes in NtJIH1 and NtCYP450 family were closely related to tobacco resistance to insects.2.Overexpression and knockout of NtJIH1 and NtCYP450Through CRISPR/Cas9 and transgenic overexpression technology,NtJIH1-1,NtJIH1-2,NtCYP450-Target1 and NtCYP450-Target2 knockout mutant plants,VIINtCYP450-2 and NtJIH1-1 offspring transgenic overexpression plants were obtained.Currently,we obtained homozygous mutants of NtJIH1 and T1 generation of mutation plants of the common target site for NtCYP450.The mutation forms of these mutant plants are mainly based on insertion and deletion.The sequencing results of PCR amplification of CDS for NtJIH1-1 and NtJIH1-2 showed that the expression level of NtJIH1-2 gene was very low,which was about one-seventh of NtJIH1-1.The expression of defense-related genes in mutants were detected by qRT-PCR(Quantitative Real-time PCR).The results showed that,except for the homozygous mutant plants of NtJIH1-2,most of the defense-related genes of the mutants were significantly up-regulated.These genes included JA signal pathway marker gene PR1b(pathogenesis-related protein 1b precursor),SA signal pathway marker gene PR1a/c(pathogenesis-related protein 1 a/c),PR2(pathogenesis-related protein 2)and CHN50(endochitinase B),Eth signaling pathway gene marker genes ACC OXidase and EFE26(ethylene-forming enzyme),HR signaling pathway marker gene HSP201(heat shock protein)and HIN1(Harpin-induced gene 1)and ROS signaling pathway marker gene GST1(Glutathione S-transferase).3.Detection of insect resistance and resistance indicators of NtJIH1 and NtCYP450knock-out mutant linesThe NtJIH1 and NtCYP450 knock-out mutant lines obtained by expanded culture were applied with cotton bollworm and aphids,respectively,to detect the defense ability.The results showed that,the body weight of cotton bollworm feeding on the T1 and T2 generations of Nt JIH1-1 mutants decreased about 40% and 60%,respectively,after 4days of feeding compared with wild-type plants.The results also showed that the content of plant trypsin inhibitor and callose increased significantly compared with the control plants,indicating that the development of this resistance may be related to increasement of plant trypsin inhibitor and callose.Adding aphids to NtJIH1 mutants showed the numbers of progeny of aphids fed on the NtJIH1-1 mutants did not change significantly after 10 days.Similar insect-resistant assay show that NtJIH1-2 mutants did not show obviously enhanced insect resistance.The sequence identity between is NtJIH1-1 and NtJIH1-2 is about 96%.NtJIH1-1 genes were derived from the diploid ancestor species S genome(Nicotiana sylvestris),while NtJIH1-2 is from the diploid ancestor species T genome(Nicotiana tomentosoformis).NtJIH1-1 and NtJIH1-2 are orthologous genes.However,the expression level of NtJIH1-2 gene was very low,which was about one-seventh of NtJIH1-1.These two genes might undergonefunctionally selective evolution during the reorganization of heterotetraploid tobacco.In addition,we found that the size of homozygous mutant plants of NtJIH1-1 is significantly smaller than the control plants.Similarly,the resistance of NtCYP450 mutants were tested by infesting cotton bollworm and aphids.The results showed that,the body weight of cotton bollworm feeding on the T1 and T2 generations of NtCYP450 mutants decreased about 49.5% and24%,respectively,after 4 days of feeding compared with wild-type plants.The results also showed that the content of callose increased significantly compared with the control plants,indicating that the development of this resistance may be related to increasement of callose.Aphids were added to Nt CYP450 mutants for insect-resistant assay.The results showed that,compared with wild-type plants,the numbers of progeny of aphids fed T1 and T2 generations of NtCYP450 mutants were decreased by 57% and46% after 10 days,respectively.The results also showed that the content of ethylene increased significantly compared with the control plants,indicating that the development of this resistance may be related to increasement of ethylene.In this study,mutants of NtJIH1-1,NtJIH1-2,NtCYP450-Target1 and NtCYP450-Target2 were obtained through CRISPR/Cas9 technology.The insect bioassays showed that KO-NtJIH1-1 plants showed significantly enhanced resistance to cotton bollworm,while KO-NtCYP450-Target1 and KO-Nt CYP450-Target2 plants showed significantly enhanced resistance to both cotton bollworm and aphids. |
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