| All Panax family plants possess high medical values and the main active components are ginseng saponins.Panax japonicus C.A.Mey.belongs to Panax family and its content of total saponins is the highest among the family.The total saponins content in root is more than 15%,which are 2-7 seven times compared to those in P.ginseng and 3 times to those in P.quinguefolium.Ginseng saponins are triterpenoids produced in plant secondary metabolism.Triterpenoids are formed via various cyclations of squalene.?-amyrin synthase(?AS)and dammarenediol-II synthase(DS)are the key enzymes in the triterpenoids biosynthesis.In the research,?AS and DS genes were cloned from P.japonicus with PT-PCR and rapid amplification of cDNA ends(RACE).The sequence analysis,function prediction and prokaryotic expression were also conducted.The gene sequences were optimized,inserted to the dual T-DNA vector and transferred to rice "Taijing 9" and“Shuhui 527”.The transgenic rice plants were assayed and analyzed.Thus,this study has originally produced the new germplasms of "ginseng rice" with functional component saponins.The main experiment results are as follows:1.Cloning and homology comparison of the ?AS gene.This research cloned the full length cDNA of ?AS from the roots of P.japonicus.The gene,named with Pj?AS in our study,is 2715bp in length with an ORF of 2286bp,codes 761 amino acids and the theoretical molecular weight is 87.8kDa.In the DNA sequence,the Pj?AS shows 96%?83%?81%?80%homology with ?AS2 of P.ginseng(Korea),?AS of Aralia elata,?AS of Betula platyphylla and ?AS of Malus x domestica,respectively.Whereas in the amino acid sequence,Pj(3AS shows 98%,86%,85%,83%identity and 98%,91%,92%,91%similarity,when compared with the ?AS2 of P.ginseng(Korea),?AS of Aralia elata,?AS of Betula platyphylla and ?AS of Malus x domestica,respectively.2.Cloning and homology comparison of DS gene.This research cloned the full length cDNA of DS gene from the roots of P.japonicus.The gene,named with PjDS in our study,is 2572bp in length with an ORF of 2310bp,codes 769 amino acids and the theoretical molecular weight is 88.2kDa.In the DNA sequence,the PjDS shows 98%?98%?97%?97%homology with DS of P.ginseng(Korea),P.quinguefolium,P.notoginseng and ?AS(AB 122080.1)of P.ginseng(Korea),respectively.Whereas in the amino acid sequence,PjDS shows 99%,99%,98%identity and 99%,100%,99%similarity,when compared with the DSs of the P.ginseng(Korea),P.quinguefolium and P.notoginseng,respectively.3.Function prediction and prokaryotic expression of ?AS and DS.The analysis result of the conserved domain search of the deduced amino acid sequence indicated that Pj?AS and PjDS contained the conserved domains of class ? terpene cyclases and protein prenyltransferases beta subunit.Analyzing result in the blocks database of the deduced amino acid sequence indicated that Pj?AS and PjDS contained the 6 functional domains of the terpene synthase family and the functional domains of the prenyltransferase/squalene oxidase.Based on the above results it could be inferred that Pjf?AS and PjDS had functions of ?-amyrin synthase gene and dammarenediol-?synthase gene,respectively.4.Optimization of the target genes and gene synthesis.The coding sequences of the ?AS and DS genes were codon optimized according to the codon preference of rice.The signal peptide sequence of the glutelin gene was added to the 5' of the target gene,and the ER retention signal sequence of the glutelin gene was added to the 3' of the target gene.The optimized target genes were named SPAS(2382bp)and SPDS(2406bp),respectively,and sent to synthesis.In the optimized gene sequences,the rice utilized the rare codons(less than 10%)less frequently and the preferential codons more frequently.5.Construction of the dual T-DNA expression plasmid and transformation to rice.The dual T-DNA plant expression plasmids,pCDMAR-SPAS-hpt,pCDMAR-OPAS-hpt(without signal peptide sequence and ER retention signal sequence of the glutelin gene)and pCDMAR-AS-hpt,pCDMAR-SPDS-hpt were constructed.The four plasmids were transferred to the rice variety "Taijing 9" or" Shuhui 527" respectively with agrobacterium-mediated transformation.6.Rice genetic transformation and detection.(1)The PCR detection of the transgenic plants.The PCR amplification the hygromycin resistance gene HPT showed that all of the obtained plants were positive.The PCR amplification of the target genes showed that the number of the positive plants of SPAS transformed 'Taijing 9' was 38(positive rate 58.5%);OPAS transformed 'Taijing 9' was 14(positive rate 24.6%);?AS transformed 'Taijing 9'Tand"Shuhui 527" were 12(16.4%)and 5(11.4%)respectively;SPDS transformed'Taijing 9' and "Shuhui 527" were 10(13.7%)and 10(15.6%)respectively.(2)hiTAIL-PCR analysis of the insertion site in the T-DNA region.hiTAIL-PCR was used to amplify the flanking genomic sequence of the target gene.The results showed that SPAS and SPDS etc.were inserted to the genomes of the rice plants respectively.The flanking genomic sequences of the T-DNA region in the positive plants,SA30,SA37,SA43,7D6,7D22,7D24 and 7D36,were obtained.Homology compared and BLAST search of the resulting flanking sequences were conducted by referencing the genomic sequences of the rice varieties '9311' or Nipponbare.The result showed that the insertion site of the SPAS in the plant SA30was at 9309.76kb of the chromosome No.5;that of the SA37was at 5301.91kb of the chromosome No.12;those of the SA43 were at 31969.15kb and 31972.45kb of the chromosome No.2;the insertion sites of the SPDS in the plants 7D6,7D22,7D24 and 7D36 were all at 31741.45kb of the chromosome No.3.(3)qRT-PCR analysis of the target gene in the transgenic rice plants.The relative expressions(RQ)of the target gene in the transgenic rice were as follows:SPAS 2658.8-13915.9,OPAS 320.3-1544.9,?AS 3329.1-17777.4 and SPDS 5589.2?6399.7,with the negative plants set to 1.It showed that ? the expression of the OPAS was significantly lower than that of the ?AS,whereas the expression of the SPAS is comparable to that of the ?AS.This suggested that the optimization of the ?AS could not improve its expression in the transgenic rice but lower the expression instead.The optimization of the non-coding region(signal peptide sequence and ER retention signal sequence of the glutelin gene)increased the expression in the transgenic rice significantly.? SPDS showed insignificant difference among different transgenic plants.(4)Western blot analysis of the target protein in the transgenic rice.The?-amyrin and dammarenediol-II synthase encoded by the Pj?AS and PjDS of P.japonicus respectively,were detected in the transgenic rice plants,the molecular weights of both proteins were about 88kDa,which was consistent with the expectation,while the negative plants showed no bands in the Western blot analysis.(5)HPLC assay of the saponins content in the transgenic rice.The reflux extraction was utilized to extract the saponins in the transgenic rice plants.The HPLC analysis showed that the target products PPD and PPT could be detected in all transgenic plants of SPDS except 7D34.The PPD content was 0.06‰-0.20‰ and the PPT content was 0.00‰-1.73‰(the PPT content in 7D34 was 0).In the transgenic plants of SPAS,OPAS or ?AS,the target product Ole could be detected in only few plants,with content 0.01‰-0.13‰.Except for AS60,the Ole contents in all other plants detected were less than 0.10‰?... |
PDF Full Text Request