Study On Blast Resistance Of Transgenic Rice Transformed By Stress Resistance Related TAC Clones From Oryza Officinalis Wall

In this study,the cultivated rice materials obtained from the transformation of the anti-inverse related TAC Clones from Oryza officinalis Wall by the South China Agricultural University with Oryza sativa L.japonica.cv.Nipponbare as the receptor were used.The materials included:1.High generation?T10?TDF series of stable strains and low generation?T3-T5?TR series of materials cloned by transforming random TAC;2.Transformation of TAC clones of high-generation?T10?D series stable strains based on DREB conservative sequence screening;3.Transformation of the low generation?T3-T5?NBS-LRR,STK and NAC series materials obtained by TAC clones based on the sequence screening of NBS-LRR,NBS-LRR and NAC conservative domains,respectively.Identification of molecular markers of transformed TAC clones,resistance identification of leaf blast strains and genetic analysis of resistance was done in order to clarify the leaf blast resistance level and genetic basis of the resistance the transformed lines,further explore the functions of the resistant genes and lay a foundation for the breeding of resistant varieties to leaf blast.The main findings are as follows:By the artificial inoculation of D series strains with leaf blast,30 out of 55 D series strains were identified to be resistant,including 14 broad-spectrum strains with higher resistance levels with a spectrum ratio of 65.4%⁸6.7%,accounting for46.67%of the total number of identified plants.16 moderately resistant strains had a spectrum ratio between 42.6 to 63%.26 D series strains were inoculated with artificial leaf blast,8 strains had a broad spectrum range with ratio between 66.7%and 85.7%.There was strong resistance to leaf blast with a medium spectrum ratio between 40 to 60%among 4 moderately resistant strains.D40 strains showed the weakest resistance to leaf blast.14 Strains of D series identified with strong resistance to the leaf blast disease were naturally induced in field beds.6 strains showed leaf blast resistance between grades 0-2.Thirteen strains showed grade 0-2resistance to leaf blast,among which d4-2 LTH showed high resistance to leaf blast,grade 0 resistance to leaf blast.Fifteen?15?TDF series strains were resistant to 55strains,including 6 broad spectrum strains which were highly resistant to leaf blast and 9 moderately resistant materials,which accounted for 65.67%of the total number of identified strains.However,TDF12 strains were weak.There were differences in the resistance of TDF strains to the strains inoculated with leaf blast,among which 7 strains were fully resistant to GD09-3 and 5 strains were fully resistant to HB6.GW3,FJ3 and SH4 showed partial resistance,and resistance/total plant number was 0.71.There were differences in the resistance of NBS-LRR series strains to the 8 leaf blast strains,of which 112 strains had the strongest resistance to HB1 strains with an average level of disease resistance of 1.83 strains showed high resistance to GD09-12 strains,and the strains of GW6,HB5,HB6,Jj01-1 and FJ10showed resistance to leaf blast,and resistance total number of plants?0.75.STK series of GW6,GW3,HB4,FJ10 strains showed high resistance to leaf blast,resistance/total plant number ratio was between 0.54-0.83,but the jj01-1 strain was not resistant as all were susceptible.NAC series strains of GW6 and GD09-12 strains had strong resistance to leaf blast,resistance/total plant number>0.79.HB8,HB4 and HB6 strains were resistant.In addition,the results of current study found that more susceptible strains were inoculated to HB5,jj01-1,HB1 and FJ10 strains,and the resistance/total number of strains was small.The resistance detection of 30 D series and 12 TDF series was carried out by using the HPT and SacB resistance marker genes carried by TAC vector,which could amplify the target gene band carried by the target TAC vector.Sixteen?16?out of 35 T3 generations NBS-LRR series bands were amplified into target bands,92 out of 100 T4 generation plants were amplified into target bands.Seventy-four?74?homozygous lines and 182 T5 generation plants were screened,170 of which were amplified into target bands,and 159 homozygous lines were screened out.15 out of76 T3 generation series strains were amplified into target bands.Among 45 T4 plants,37 were amplified into target bands,with 11 plants being homozygous lines.Out of54 T5 generation plants,45 were amplified into target bands with 39 homozygous lines screened.13 T3 generation NAC series strains were amplified into target bands out of a total of 38 strains.45 T4 generation plants were able to amplify target bands,and 45 homozygous lines were found.36 strains T5 generation plants,34 strains amplified target bands,31 homozygous strains were screened,indicating that 5selected medicinal wild rice TAC cloned and transformed rice strains based on transcription factor conservative domain sequence screening,selected from the homozygous and disease-resistant strains T3 to T5 gradually increased.The T5generation basically achieved homozygosity with all showed better and stable resistance to the disease.The number of target genes was gradually increased,and the transcription factors of DREB,STK,NBS-LRR and NAC were successfully transferred to these transgenic strains respectively,which may be used as positive regulatory factors to participate in the broad-spectrum disease resistance effect of transgenic strains,and to promote their ability of enhanced resistance to leaf blast.The high-generation stable disease-resistant transgenic strains D1,D4-1,D4-2and D38 were selected and combined with LTH to identify F₂.D1 LTH and d4-1LTH were inoculated with HB5 strain,and D4-1 LTH and D4-2 LTH were inoculated with HB8 strain.The resistance to leaf blast was 3:1,indicating that the resistance of D1,D4-1 and D4-2 to leaf blast was controlled by 1 pair of dominant genes or dominant QTL loci.D1 LTH was inoculated with HB8,and the resistance to leaf blast was in line with 15:1,indicating that the resistance of D1 to HB8 was controlled by 2 pairs of dominant genes.D4-2 LTH was inoculated with HB5 strain.D38 LTH was inoculated with HB5 and HB8 strains respectively,and the anti-inductive separation ratio was consistent with 9:7.This indicated that D4-2 pair of HB5 strains,D38 pair of HB5 and HB8 strains of leaf blast resistance was related to the control of two pairs of interacting genes.D1?LTH,D4-1 LTH,D4-2?LTH,and D38 LTH were inoculated with HB5 and HB8 strains,and the susceptibility separation ratio of leaf blast was in line with 3:1,indicating that the resistance of D1,D4-1,D4-2,and D38 to leaf blast was controlled by 1 pair of dominant genes or dominant QTL loci.D1 LTH,d4-2 LTH and D38 LTH can all specifically amplify the target TAC to clone the HPT target gene bands,and the anti-sensitivity separation ratio is in line with 9:7,but there is no ideal resistance separation ratio for l4-1 LTH,indicating that the disease resistance of D1?LTH,D4-2?LTH and D38LTH may be controlled by two pairs of complementary genes.