Silencing Of AtCDPK1 And AtCDPK1α In Arabidopsis Thaliana By RNA Interference

Calcium-dependent protein kinases (CDPKs) belong to an important protein kinase family, and play critical roles in plant responses to biotic and abiotic stress. Among the 34 CDPK members encoded in Arabidopsis genome, AtCDPK1 and AtCDPK1 share high homology. Existing research results showed that these two CDPKs also have similar functions. Take advantages of RNAi technology in specificity and robust on gene family members, it was used in this study to silence AtCDPK1 and AtCDPK1a. The results showed:1 Totally five RNAi constructs were made. Three constructs were designed to target to different regions of both AtCDPK1 and AtCDPK1a, and two constructs were targeted to AtCDPK1 and AtCDPK1a respectively. The above mentioned constructs were transformed into Arabidopsis Columbia ecotype by flower-dipping method mediated by agrobacteria. Two co-supression constructs and the one targeted to AtCDPK1a had obtained homozygous transgenic lines, the other two constructs are screening the T2 transgenic plants.2 Using semi-quantitative RT-PCR, the degree of gene silencing in homozygous transgenic plants were checked:(1) For the co-supression construct CDPK-a, all the six transgenic lines showed high level of silencing on both genes. Among them, line 10-2 showed strongest level of silencing.(2) For the co-supression construct CDPK-c, silencing level of AtCDPK1a was stronger than AtCDPK1 in all five lines. Among them, line 2-2 has the strongest level silencing.(3) Nine lines of CDPK1a-2 construct targeting to AtCDPK1a showed specific silecing of the target gene. Lines 29-4 and 36-5 showed much stronger degree of silencing.3 High specificity of silencing was confirmed by semi-quantitative RT-PCR in transgenic homozygous CDPK1a-2 lines.