Cloning And Transformation Of Several Fruit Ripening-related Genes From Papaya (Carica Papaya L.)

Papaya, Carica papaya, a member of the family Caricaceae, is one of the most important tropical and subtropical fruit trees. Like most climacteric fruit, papaya ripens quickly after harvest and then soften and deteriorate rapidly. It is also sensitive to low temperature and cannot be stored in cool conditions. Hence, large losses during the storage and transportation of papaya are caused. Up to 23.7% of the harvest is lost in transit from the farmer to the consumer. With the molecular biology development, genetic engineering was used to modify genes in order to regulate fruit ripening. Studies of cloning and transformation of fruit ripening-related genes were rare and the reports of genetically modified varieties on sale were not seen. In order to know more about molecular mechanism of fruit ripening and to seek more effective anti-softening genes, cDNA-AFLP technique was used to identify differential gene expression during papaya fruit ripening and some of the differential expression genes were cloned by RACE. Pectate lyase gene was cloned too. PL antisense expression vector and PL andβ-Gal antisense expression vector were constructed and transformed into papaya mediated by Agrobacterium tumefaciens co-transformation, and the kan-resistant somatic embryo were obtained. The major results obtained from the study are summarized as follow:1 Studies on differential gene expression of papaya fruit at different ripening stages by means of cDNA-AFLP techniqueFifty TDFs were obtained from the analysis of color break and half yellow stage of papaya fruit by cDNA-AFLP. Bioinformatics analysis showed that twenty eight TDFs were homologous to known function genes, five TDFs were homologous to uncharacterized genes, while seventeen TDFs did not show significant matches to any genes in the Genbank database. Twenty eight genes of known function TDFs could be divided into different functional groups including gene expression regulation, DNA and protein synthesis and transport, protein degradation, energy metabolism, nutrient metabolism and stress responding. Among the eleven expression regulation genes, seven was signal transduction genes, three was transcription factors, and one was post-transcriptional regulation gene. These results indicated that fruit ripening and senescence is a complicate physiological and biochemical processes and should be regulated by a complex system of signal induction and physi-biochemical metablism.2 Cloning and analysis of differential expression of fruit ripening genes and Actin gene from papayaFive differential expression genes and one Actin gene were cloned by rapid amplification of cDNA ends (RACE) from papaya. The gene expression patterns in papaya fruit at different ripening stages was analyzed by semi-quantitative RT-PCR.The full length cDNA of CpPOD was 1124 bp. Sequence analysis showed that the start codon was could not find, this may be caused by mistaken sequencing or base losting. Semi-quantitative RT-PCR analysis showed that the expression of CpPOD was highest in color break stage and decreased when fruit began to ripen and soften.A full-length cDNA clone encoding MYB was cloned from papaya. The full length cDNA of CpMYB was 1124 bp with an opening reading frame of 879 bp encoding a protein with 292 amino acids. Semi-quantitative RT-PCR analysis showed that the expression of CpMYB was increased gradually with papaya fruit maturing and decreased when fruit began to senesce.GDP-mannose pyrophosphorylase gene was cloned from papaya. The full length cDNA of CpGMP was 1544 bp. Open reading frame which encoded 361 amino acids was cloned from genome DNA and cDNA. ORF of DNA sequence was 1775 bp which including 4 extrons and 3 introns. Semi-quantitative RT-PCR analysis showed that the expression of CpGMP increased gradually with papaya fruit maturing and decreased when fruit began to soften.Proteasome subunit alpha type-6 gene was cloned from papaya. The full length cDNA of CpPAA1 was 1025 bp with an ORF of 741 bp encoding a protein with 246 amino acids. Semi-quantitative RT-PCR analysis showed that the expression of CpPAA1 increased gradually with papaya fruit maturing and decreased when fruit began to senesce.CpSRF3 partial sequence was obtained by 3'RACE which encoded 128 amino acids. Gene domain analysis showed that CpSRF3 is a kind of receptor-like protein kinases with a leucine-rich repeat. Semi-quantitative RT-PCR analysis showed that the expression of CpSRF3 decreased gradually with papaya fruit ripening and senescence. A full-length cDNA clone encoding Actin was amplified from papaya. Sequence analysis showed that it contained a complete ORF of 1134 bp coding for 377 amino acids. Actin gene was often used as an internal standard in gene expression analysis.3 Cloning of pectate lyase gene from papaya and construction of antisense expression vectorComplete 3' and part 5' terminal sequence of PL gene were obtained by RACE from papaya. Using two specific primers, open reading frame of 1464 bp encoding 385 amino acids was cloned from genomic DNA. There were 4 extrons and 3 introns in the sequence. Then, antisense expression vector pBP was constructed and transformed into Agrobactrium tumefaciens EHA105.4 Construction of PL andβ-Gal antisense expression vector of papayaPL andβ-Gal conserved domains were cloned using specific primers designed according to those gene sequences. The PL andβ-Gal cDNA were inserted into the site between CaMV 35S promoter and Nos terminator of the expression vector pCAMBIA1301-35S-GUS-Nos with reverse orientation respectively. So, two antisense expression vectors pCP and pCG were obtained. Then, PL andβ-Gal antisense genes with 35S promoter and Nos terminator were inserted into expression vector pCAMBIA2301 together to generate double gene plant expression vector pCPG by different mode.5 Studies of establishment of somatic embryogenesis and transformation system of papayaThe effects of different hormones on the somatic embryogenesis from papaya immature zygotic embryo were studied. Results showed that the appropriate medium for callus induction was modified MS media supplemented with 10 mg/L 2,4-D and 2 mg/L KT and 0.5 mg/L BA and 30 g/L sucrose and 400 mg/L glutamine, the appropriate medium for somatic embryogenesis was modified MS media supplemented with 10 mg/L 2,4-D and 2 mg/L KT and 30 g/L sucrose and 400 mg/L glutamine. PL antisense expression vector and PL,β-Gal antisense expression vector were transformed into papaya mediated by Agrobacterium tumefaciens co-transformation, and the kan-resistant somatic embryo were obtained.