Cloning Of Defence Genes Induced By Powdery Mildew From Rubber Tree And Establishment Of Reproduction System Of Transgenic Rubber Tree Transformed With Hpa1xoo Gene

Rubber tree,a main producer of natural rubber,is of critical bioenergy plant,thereby playing a pivotal role in national economy.Powdery mildew,which belongs to one of world-wild three main diseases,will lead to the death of new yielding shoots,reduction of latex and no-seed phenotype,further severely affecting the plantation of rubber tree and production of natural rubber.Therefore,it is important to screen a good disease-resistance cultivar for development of rubber industry.However,little research has been done on the molecular mechanism of rubber tree resistance to powdery mildew and expression regulation of genes related to disease resistance.Thus,exploring good candidate genes resistancepowdery mildew will be of a great foundation for clarifying the interaction mechanism between rubber tree and powdery mildew and facilitate selecting new disease-resistant cultivars as combined with high-quality molecular breeding approaches.In the present study,we construct a mRNA database of rubber tree on differential-expression genes induced by O.heveae at different time points by the method of Differential Display Reverse Transcriptase Polymerase Chain Reaction(DDRT-PCR),and then each of EST short sequence harvested by function was annotated and classified.Furthermore,some of EST sequences were further assessed by qRT-PCR and completed their corresponding full-length cDNA fragments,respectively.In addition,HbFer gene,which was obtained base on its differential expression,was successfully transformed into tobacco for confirming its function.Finally,rubber tree transformation system was optimized,providing a foundation for integration of genes related to disease resistance into rubber tree genome.Our study results are followed below:1.A database on differential-expression genes induced by O.heveae at different time points by the method of differential display reverse transcriptase polymerase chain reaction(DDRT-PCR)was constructed.Finally,of more than 300 differential-expression fragments obtained,78 short sequences obtained were further assessed by PCR and reverse northern blot and then registered in NCBI,in which accession number of this database was named as LIBEST-028598.Through BLASTX program,these EST short sequences were divided into different pathways,such as cell wall and cytomembrane pathway,transcript factor and regulated protein,transmission,signal transduction,phytoalexin biosynthesis as well as other pathways related to metabolism.2.Eight significantly differential-expression EST sequences from EST database were further confirmed by qRT-PCR in RRIC52(O.heveae-resistance cultivar)and Reyan7-33-97(O.heveae-susceptible cultivar),and then four open reading frame sequences were obtained from genome DNA sequence of rubber tree through BLAST analysis and were respectively designated as Chitinase gene,Ferritin gene,WRKY transcription factor gene and Cupinl gene based on bioinformatics analysis,suggesting that these genes may be implicated in regulation of plant-defense mechanism in rubber tree.3.Ferritin gene,of which the full-length was 1027 bp,with 789 bp ORF encoding 262 amino acids,was registered in NCBI and accession number was JZ822680.Several transgenic tobacco plants transformed with Agrobacterium strain EHA105 carrying the recombinant constructs pCAMBIAl304-HbFer were obtained and further confirmed by PCR,GUS staining and southern blot.Moreover,RT-PCR for target gene was carried out for further confirming the expression levels.On the other hand,resistance test by inoculating Glomerella gossypii on leaves of transformants revealed that transformants showed disease resistance to some degrees.4.Several transgenic rubber plants transformed with Agrobacterium strain EHA105 carrying the recombinant constructs pCAMBIA2301-hpa1X00 were obtained and further confirmed by PCR,GUS staining and PCR production southern blot.Therefore,the established optimized transformation system will promote integration of genes related to disease resistance into genome of rubber tree and provide for a good genetic improvement.