Using RNAi to Study the Function of Up Regulation of EST Expression in Cotton SSH Library

doi:10.7668/hbnxb.2018.01.012

Abstract

Abstract: In order to further study the molecular mechanism of cotton somatic embryogenesis, this research used the RNAi technology, the pCRI1210 binary expression vector to construct the interference vector of cotton embryogenic callus, based on the expression vector of pBI121 provided by the State Key Laboratory of Cotton Biology, constructed 4 up-regulated expression ESTs interference vectors pCRI1210-EST (EST-418, EST-447, EST-496 and EST-653) from cotton embryogenic callus suppression subtractive library. From the high differentiation rate strain of W10 of 24 sterile CCRI in somatic embryo in seedling hypocotyl as receptor, Agrobacterium mediated transformation of pCRI1210-EST interference vector, dip the hypocotyl segments after induction medium for callus differentiation. The cDNA of callus was detected by semi quantitative RT-PCR technique. The results showed that the conversion of 4 ESTs interference vector after the embryogenic callus were detected in the carrier frame, the interference vector were successfully integrated into the genome of DNA, whereas the expression of 4 ESTs corresponding cDNA showed different degrees of reduction, the transformation of different interference vector of hypocotyl callus increased and fall. The hypocotyl recovery rate of the transformed EST-418 interference vector increased compared with that of the control. The hypocotyl recovery rate of the EST-447, EST-496 and EST-653 interference vectors decreased to varying degrees compared with the control. Therefore, it is concluded that the 4 ESTs played a different role in the process of callus differentiation. The EST-418 inhibited the somatic embryogenesis of cotton, while EST-447, EST-496 and EST-653 played an important role in the process of somatic embryogenesis in cotton, which provided technical and theoretical support for the isolation of cotton somatic embryogenesis related genes.

Key words: RNAi, SSH, EST, Callus

CLC Number:

Q78
S562.03

YIN Guo, LU Zhengying, LI Shiyun, HAN Yongliang, ZHANG Chaojun, LI Fuguang. Using RNAi to Study the Function of Up Regulation of EST Expression in Cotton SSH Library[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2018, 33(1): 71-79. doi: 10.7668/hbnxb.2018.01.012.

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