Study On Transformation Of Wheat With HMW-Glu (5+10) Genes By Biolistic Bombardment

To get high molecular weight glutenin subunit 5 and subunit 10 overexpressed wheat lines, wheat cultivar, Yumai 18, Yumai 21, Yumai 49 and Yunong 9901,were transformed with wheat high molecular weight glutenin subunit 5 and subunit 10 genes high-efficient expression vector by means of biolistic bombardment. In order to reduce transgene silencing and enhance expression of foreign gene, the two target genes were flanked with nuclear matrix attachment region, the MAR sequence. And coding sequence of KDEL, ER retention signal, was added to the end of the target genes to enhance the expression of target genes. To improve transformation of wheat immature embryo calli, the influence of several key parameters in microprojectile bombardment were studied. The results showed that under identical conditions, transformation ratio of Au dosage of 250 μg per shot was higher than that of 500 μg per shot and Au particle of 1. 0 μm in diameter was better than that of 1. 6 μm. And there was no significant difference between single shot and double shots. When the rupture pressure was 1300 psi, percentage of regenerated green spots after bombardment was much higher than that of 1100 psi. In target distance (6, 9 and 12 cm) experiment, effect of 9 cm was the best. 146 regenerated plantlings were gotten from the 4 genotypes. Among the 146 plantlings, PCR identification of subunit 5 gene indicated that 141 plantlings showed expected DNA band, but 5 plantlings didn't. The average PCR positive ratio was 96. 57%. Among 117 plantlings of Yumai 18, 114 plantlings were PCR positive, and the positive ratio was 97. 44%. 3 plantlings of Yumai 21 was positive, and 22 plantlings of 24 Yumai 49 plantlings were positive, the positive ratio was 91. 67%. There was only 1 plantling of Yunong 9901, and the PCR detection result was positive. PCR identification of subunit 10 gene indicated that 138 plantlings showed expected DNA band, and 8 plantlings didn't. The average PCR positive ratio was 94. 52%. Among 117 plantlings of Yumai 18, 112 plantlings were PCR positive, and the positive ratio was 95. 72%. All the 3 plantlings of Yumai 21 were positive, and 21 plantlings of 24 Yumai 49 plantlings were positive, the positive ratio was 87. 5%. The only plantling of Yunong 9901 was positive. In SDS-PAGE experiment of the seeds, the HMW subunit SDS-PAGE band patterns of Yumai 21, Yumai 49 and Yunong 9901 didn't show any apparent change. Among the 117 regenerated plants of Yumai 18, 7 plantlings showed seed HMW-glutenin SDS-PAGE band pattern change. Compared to wild type, there were 2 extra HMW-glutenin bands. But the position of the 2 bands was not consistent with the expected position of subunit 5 and subunit 10. And moreover, the 7 regenerated plants with changed band pattern were all in the transformants of plasmid without KDEL coding sequence. This interesting result needs to be researched further.