Abstract：The simultaneous transfer of multiple genes into plants helps researchers to improve complex traits in plants. In this study, a spacer sequence cloned from pGWB605 was inserted into pXCS-HAStrep, and the co-expression vector named pXCS-Dgene-HAStrep was constructed. HbWRKY11 and HbWRKY7 were cloned into pXCS-Dgene-HAStrep. The plasmid was then introduced into Agrobacterium strain GV3101 (pMP90RK) and transformed into WT Arabidopsis thaliana (Col-0). Eighty-four Basta resistant lines were obtained, and eight of which were randomly selected for semi-quantitative RT-PCR and qRT-PCR analysis. The results showed that HbWRKY11 and HbWRKY7 could express simultaneously, and the expression level of HbWRKY7 was higher than that of HbWRKY11, but the difference was not significant (Pr=0.4867>0.05). The different expression levels of HbWRKY11 and HbWRKY7 were observed among transgenic lines. Among 8 transgenic lines, the highest expression levels of HbWRKY11 and HbWRKY7 were detected in two single-copy T-DNA-transformed lines, S2 and S5. Four single-copy transgenic Arabidopsis lines (L3, L4, J1, J5) had been constructed by overexpressing HbWRKY11 and HbWRKY7 in previous study, respectively. T3 seedlings of L3, L4, J1, J5, S2 and S5 were investigated under cold and jasmonic acid (JA) stress. The results showed that cold- responsive AtCOR47 was strongly induced in all of six lines and wild type genotype under cold stress, and the highest expression level of AtCOR47 appeared in line J5 when all of six lines were treated under cold stress for 24 h. The increase level of AtCOR47 in four single-gene overexpressing lines was higher than that in wild type genotype under cold stress; otherwise the increase level in double-gene co-overexpressing lines was lower than that in wild type genotype. The increase level of soluble sugar content in double-gene co-overexpressing lines was also lower than other lines. Although JA-responsive AtCOI1 was induced under JA stress, the increase level of AtCOI1 in sigle-gene overexpressing lines was lower than that in wild type genotype, and the highest increase level was in double-gene co-overexpressing lines when they were treated under JA stress for 6 h. All the results indicated that two genes can express seperately under one ORF cassette in transgenic plant.
