The Analysis Of Differential MRNA For The Genomic Function Of Houttuynia Cordata Thunb. Affected By Foreign Antimicrobial Peptides

Objective:The study analysed the difference of transcriptome between GM Houttuynia Cordata Thunb.and non-GM Houttuynia Cordata Thunb. Our finding will provide some previous evidence for the judgement of the expected effect of transgenic plant, and then establishes the foundation for the safety assessment of transgenic plant to some degree.Methods:1. The total RNA from Houttuynia cordata Thunb.leaves were isolated respectively by four methods including RNAiso for Polysaccharide-rich Plant Tissue, CTAB, SDS Ⅰ and SDS Ⅱ. Six reagent combinations were selected to extract the supernatant of homogenate,including chloroform, potassium acetate+chloroform, sodium chloride+chloroform, phenol/chloroform/isoamyl alcohol(PCI), potassium acetate+PCI and sodium chloride+PCI. The purity, productivity and integrity of RNA were analyzed by UV absorbance results and agarose gel electrophoresis figures. Then RT-PCR for18S RNA were acted by PrimeScript Reverse Transcriptase and Quantscript RT kit used the total RNA by CTAB method.2. The total RNA by CTAB together with PCI from Houttuynia cordata Thunb. leaves was used to RT-PCR by PrimeScript Reverse Transcriptase and Quantscript RT kit with. Then78paris of primer with3anchor primers and26random primers was added in mRNA differential display PCR with the cDNA obtained by RT. The products were separated by agarose gel electrophoresis and polyacrylamide gel electrophoresis respectively. Polyacrylamide gels were colorate by silver staining.3. The mRNA differential display RT-PCR were acted on GM Houttuynia Cordata Thunb. and non-GM Houttuynia Cordata Thunb. which were same in growing condition, grow status and growth phase.Results:1.The purity and integrity of total RNA from Houttuynia cordata Thunb. leaves were fine by RNAiso for Polysaccharide-rich Plant Tissue, CTAB and SDS Ⅱ,when PCI or PCI together with NaCl were used in the extraction. The productivity was maximum with CTAB. The results of RT-PCR for18S RNA all had clear bands.2. The results were divese partly with cDNA produced by PrimeScript Reverse Transcriptase and Quantscript RT kit respectively when the same primer pair was used. The bands were more distinct and clear in the photographs of polyacrylamide gel electrophoresis with silver staining than that in the agarose gel electrophoresis photographs.3. The results from mRNA differential display RT-PCR with GM Houttuynia Cordata Thunb.and non-GM Houttuynia Cordata Thunb. suggested:the polyacrylamide gel electrophoresis photographs showed44differential bands,when PrimeScript Reverse Transcriptase was used in RT; while the photographs showed142differential bands,when Quantscript RT kit was used in RT.Conclusion:1. CTAB together with PCI was condidered the appropriate method to isolate the total RNA from Houttuynia cordata Thunb. leaves, after the extraction methods were optimized and RT-PCR were acted. The isolated RNA by CTAB together with PCI had better purity, integrity and productivity, which were able to reverse transcriptase and were amenable to the study of DDRT-PCR. The results of RT-PCR indicated the two methods of RT were effective including PrimeScript Reverse Transcriptase and Quantscript RT kit.2. The system were builded for DDRT-PCR of Houttuynia cordata Thunb. leaves,after the programs were designed involving the RNA isolation, reverse transcription,PCR and the results display. The results from two methods of reverse transcriptase were diverse that suggested the method of reverse transcriptase was an important factor to affect DDRT-PCR. They were comprehensive with supplement each other when the differential bands were analysed. The results were fine after PCR products were separated by polyacrylamide gel electrophoresis and were colorate by silver staining, which had better clatity and sensitivity than agarose gel electrophoresis. Moreover, the operation was simple and quick.3.The mRNA of GM Houttuynia Cordata Thunb.and non-GM Houttuynia Cordata Thunb. had some differences by DDRT-PCR. Which indicated the Genome function of GM Houttuynia Cordata Thunb. were affected by foreign antimicrobial peptides in a certain degree. The influence will produce varies in the expression of protein and the synthetize of metabolites. The study can supply integrant information and basis for the detection of the non-extended effect and the futher investigation of safety assessment with GM Houttuynia Cordata Thunb.