Identification And Genetic Transformation Of Citrus MAPK Genes And PR Genes Induced By Huanglongbing

Citrus is the most important fruit crop in the world,with an annual production of more than 110 million tons distributed in about 140 countries,mainly in tropical and subtropical areas.China is the first producer and the major consumer for the fresh citrus fruit market,with a production of almost 36 million tons and acreage of more than 26.0 million hectares in 2016.However,Citrus Huanglongbing(HLB),caused by Candidatus liberibacter asiaticus(CLas),is the most devastating disease of citrus for which no cure has been found.It is spread by citrus psyllids.Almost all known citrus varieties including those from their closely related genera can be infected.The typical symptoms of HLB disease are blotchy mottling of leaves often resembling zinc deficiency symptoms which leads to the typical appearance of yellowing of shoots in the tree.Additionally,abnormal coloration and small misshapen fruits with aborted seeds is characteristics of HLB-infected trees.At present,the combined program including using virus-free nucellar seedlings of citrus,rooting out the trees that carrying Candidatus liberibacter asiaticus(CLas),using pesticides and other chemicals to exterminate Asian psyllid,has been used for control of citrus huanglongbing in the field.Use of a large number of chemical pesticide is one of the most effective control measures for the psyllid,which brings pressure to the ecology environment.The dilemma for citrus breeders is there are only a few citrus cultivars and resources show tolerance but not significant resistance to HLB.It is almost impossible to breed the HLB-resistant descendants by using the traditional cross-breeding.Therefore,the anti-disease transgenic breeding maybe an effective treatment to acquire the HLB-resistant citrus by transferring the genes response to HLB attack.During co-evolution with pathogens,plants have developed a set of defense strategies to cope with biotic stress,with an innate immune machinery consisting of pathogen-associated molecular patterns(PAMPs)-triggered immunity(PIT)and Effector-triggered immunity(ETI).Plant trigger PTI when the pathogen attacking,a few pathogen secrete effectors to inhibit PTI and activate MAPK cascade,then a sequence of PR genes was rapidly activated,the plant evolves the corresponding specific R protein and trigger ETI.Mitogen-activated protein kinase(MAPK)cascade reaction is composed of three protein kinase MAPKKK-MAKK-MAPK,and MAPK is the terminal signal of cascade reaction.Pathogenesis-related proteins(PRP)play important roles in the process of plant disease resistance.In recent years,there are many reports about ransformation of MAPK genes and PR genes to increase the plants disease resistanc.However,the expression pattern of MAPK genes and PR genes response to citrus HLB needs to be explored in depth.Whether over-expression the MAPK genes and PR genes that response to HLB infection can improve the citrus HLB resistance will be involved in this dissertation.In this study,we focused on MAPK genes and PR genes as candidates to screen the genes that induced expression to citrus Huanglongbing.Egyptian orange was used for the genetic transformation.Over-expressing vectors were constructed and transgenic transferred into agrobacterium EHA105.The transgenic plants were obtained in this study.The results are listed as follows:1.Screening of HLB plants and non-HLB plants.13 leaf samples of the Citrus Sinensis were collected from the nursery,HLB plants were identified by PCR using the HLB-specific primers.4 HLB plants and 4 non-HLB plants were selected as materials in the subsequent experiments.2.Primer design.The citrus genome sequence data and EST data on the NCBI were downloaded and used to design primers for clone from a citrus c DNA library.15 MAPK genes,including Cs MAP,Cs MAP1,Cs MAP4-like,Cs MAP7,Cs MAP7-like,Cs MAP9,Cs MAP9-like,Cs MAP13-like,Cs MAP15,Cs MAP19-like,Cs MAP20-like,Cs MAPMMK1,Cs MAPMMK2,Cs MAPNTF3 and Cs MAPKB1,17 PR genes,including Cs PR1,Cs PR2,Cs PR3,Cs PR4,Cs PR5,Cs PR6,Cs PR7,Cs PR8,Cs PR9,Cs PR10,Cs PR11,Cs PR12,Cs PR13,Cs PR14,Cs PR15,Cs PR16 and Cs PR 17,were cloned.3.Screening the genes response to citrus HLB.The total RNA was extracted from HLB plants and non-HLB plant leaves.c DNA was synthetized by using the reverse transcription kit.The expression level of all genes in HLB plants and non-HLB plant was measured by real-time PCR.The results showed the relative expression level of Cs MAP9 and Cs MAP9-like in HLB plants was 1.7 and 1.8 times than in non-HLB plants,respectively.Two PR genes expression in HLB infected plants were increased significantly,the expression of Cs PR12 was 2.9 over-expression line and Cs PR14 was about 7.7 over-expression line.The expression levels of the other genes were not increased significantly.Compared to non-HLB citrus plants,the relative expression level of Cs PR12 and Cs PR14 was up-regulated 1.9 and 6.7 times in HLB citrus plants by Candidatus liberibacter asiaticus(CLas),respectively.In conclusion,Cs MAP9,Cs MAP9-like,Cs PR12 and Cs PR14,were significantly up-regulated express by HLB,this indicates that the four genes may play important roles in the resistance to HLB.We screened Cs MAP9,Cs MAP9-like,Cs PR12 and Cs PR14 for genetic transformation.4.Cloning and transformation of candidate genes.The over-expression vector of Cs MAP9,Cs MAP9-like,Cs PR12 and Cs PR14 were constructed by PCR-enzyme connection method.The gene CDS fragments were directly cloned into p FGC5941 vector by using restriction enzyme cleavage and connection.The recombinant plasmid was transferred into Agrobacterium tumefaciens EHA105 by using freezing and thawing method.The genetic transformation of citrus was operated by using Agrobacterium-mediated method.5.Identification of transgenic plats.PCR amplification was performed by the using the primer designed baced on nti-Basta marker gene sequence on the p FGC5941 vector.A total of 12 Cs MAP9 transgenic plants,15 Cs MAP9-like transgenic plants,11 Cs PR12 transgenic plants and 14 Cs PR14 transgenic plants were obtained.The expression of Cs MAP9,Cs MAP9-like,Cs PR12 and Cs PR14 was detected in candidate positive plants by q RT-PCR.The result showed Cs MAP9-2 and Cs MAP9-25,Cs MAP9-like-8 ?Cs MAP9-like-11 and MAP9-like-22,Cs PR12-15 and Cs PR12-26,Cs PR14-17 and Cs PR14-26 were over-expression transgenic plants.The transgenic seedlings were grafted on rootstocks and would be important citrus plant material for studying their function in HLB resistance.