Co-transformation And Identification Of Wheat With Scab Resistance-related RNAi Fragments Mediated By Gene Gun

Wheat is one of the main food sources of human beings.It is of great significance to ensure the high quality and high yield of wheat for the survival and development of human race and to maintain world peace.However,wheat production is compromised by many factors especially diseases and insect pests.Among diseases,FHB or wheat scab is common throughout the world,which reduces wheat yield and damages the quality also.The development of resistant varieties against FHB is hard through conventional breeding due to non-availability of completely resistant germplasm in wheat.Therefore,transgenic technology is used to import the disease resistant genes of other sources into the wheat,the method of preparing resistant plants by introduction of respective pathogen genes-opens new avenues to develop resistant varieties against wheat scab.In this study,to introduce multiple RNAi fragments constructs from Fusarium genes into a single wheat plant using gen-gun,to standardize the tissue culture technique to obtain transgenic wheat,to screen transgenic wheat to get homozygous wheat lines.RNAi fragments were selected and joined in different combinations from following essential genes,Cyp51 gene related to ergosterol synthesis,chitin synthaseChs3 b gene related to chitin synthesis,?-glucan synthase-Gls gene related to generation of ?-glucan,myo-inositol 1-phosphate synthase-Myo gene related to cell division and microRNAs-8mR7,8mR12-47 genes related to toxin synthesis.Expression vectors having multiple RNAi fragments were bombarded by gene gun to transform different wheat varieties including Xiangmai 76(X76),Zhengmai 9023(Z9023)and Yangmai 158(Y158).Phospho mannose isomerase(Pmi)was used as a positive screen marker for transgenic plants during tissue culture.The regenerated transgenic plants were screened out by polymerase chain reaction(PCR).Simultaneously,transgenic wheat lines obtained by other group members were also reproduced and screened.The main results are as follows: 1.Self-prepared transgenic lines:(1)Vectors expressing RNAi-fragments of genes,cyp51 BAC,gls2-gls6,and Pmi were constructed and transferred into X76.We screened and obtained one transgenic wheat line in T0,and numbered that as XII.PCR showed that XII in T1~T4 generation contained cyp51 BAC,gls2-gls6 and Pmi contructs.The foreign genes in the T4 generation didn't separate,and XII is in the homozygous generation.(2)We transformed X76 with the RNAi fragments of Gls2-t-Gls6 and chs3b-tAS2-AS3,along with the marker gene Pmi by gene gun,and 24 positive plants were screened out as positive by PCR in T0.6 of them contained all foreign fragments,13 of them contained only Gls2-t-Gls6 and Pmi,and 5 of them contained only Gls2-tGls6 gene constructs.PCR identification showed that the progenies of the 12 T0 generation lines are still separated in the T3 generation.The other 12 T0 generation lines did not detect exogenous genes in T1 generation.(3)The RNAi fragments of chs3b-AS5-t-AS5 and chs3b-AS3-t-AS3 were transferred into Z9023 and X76 by gene gun,and 11 positive plants were obtained using mannose as the screening agent.2 of them belong to Z9023 variety,contained both Pmi and chs3b-AS3-t-AS3 were identified in T0,of which the T1 and T2 generation are still separated.The remaining 9 lines belong to X76,5 of them contained both Pmi and chs3b-AS3-t-AS3,and they are still separated in T1~T3;4 of them contained only marker gene.(4)The RNAi fragments of Myo2-AS6,under the control of different promoters and Pmi were transferred into X76 by gene gun,and 5 positive plants containing both target and marker gene were screened out in T0.The PCR identification of the progeny plants showed that descendants of 4 of the 5 T0 plants were separated at much higher rate,while we observed slight separation of RNAi fragments into 1 line that entered into a homozygous phase in the T3 generation.(5)The RNAi fragment of 8mR7,activated by different promoters,and the marker gene Pmi were transferred into X76 and Z9023 by gene gun.16 lines containing 8mR7 and Pmi genes were obtained.15 of them belong to X76,and the PCR analysis showed very high separation of the target fragments in the T1 and T2 generation which decreased from generation to generation.The other 1 line belongs to Z9023 and has lower separation ratio both in T1 and T2 generations.(6)The RNAi fragments of 8mR12-47,under control of different promoters and Pmi were transferred Pmi into X76.3 positive plants containing 8miR12-47 and Pmi were obtained.All the exogenous genes are contained in the T1 and T2 generations.(7)The RNAi fragments chs3b-5*2,activated by different promoters,and Pmi was introduced into X76 by gene gun,and obtained 1 positive plant containing chs3b-5*2 and Pmi in T0.PCR results showed that plants have all the exogenous fragments in successive generations,and the current generation-T2 is homozygous.(8)Minimal expression cassette consisting of RNAi fragments from CYP51,D4 E,Opt1 and AFP genes was synthesized to transform Y158.In which D4 E,Opt1 and AFP genes are linked together.4 positive plants containing all fragments screened out in T0.The results of PCR showed that all transgenic plant offspring of the T1 generation contained all the target genes and marker gene,and the material have been homozygous.2.Transgenic lines transferred by other lab mates:(1)The reproduction and PCR identification of X76 transgenic lines JB,JV,M,J and Z,which were transformed with chs3bAS2-AS3,chs3bAS5-AS1 fragments are still in progress to achieve homozygosity.(2)The reproduced and identified of 3B T1 generation.The 3B line of variety X76 consists of RNAi constructs of chs3b5*2 under control of different promoters.In T1 generation,only 2 plants were positive for all fragements.(3)The reproduced and identified of 8R T1 generation.8R is Y158 variety transgenic line which was transformed with 8mR12-47 and different promoters.8R line is still separated in T1 generation.(4)The reproduced and identified of YZ T1 generation.YZ belongs to Y158,transformed with RNAi fragments from ZmGC?ACE?Ech42 genes,and Pmi.In T1,4 transgenic plants were obtained which were positive for all target constructs and YZ line have not separated in T1 generation.