Red-fleshed papaya is a good material to study the different carotenoids accumulation mechanism in the peel and flesh. Although the peel and flesh of papaya closely integrated into one body, the flesh coloration changing from white to red, while the exocarp coloration changing from green to yellow. In this study, the major carotenoids accumulation and the expression patterns of key carotenoid biosynthesis pathway genes in the process of papaya fruit ripening were studied, and the carotenoid biosynthetic pathways in the yellow peel and red flesh of papaya were investigated. The carotenoid composition in papaya flesh and peel were different. The major carotenoids were lutein and β-carotene in the peel, while lycopene in the flesh. The accumulation of carotenoids, including lycopene, β-carotene, and β-cryptoxanthin were considered to cause the orange-red color of papaya cv. ‘Daqing No.10’ flesh. The color of peel changed from green to yellow because of the fast degradation of chlorophyll and the appearance of carotenoids such as lutein and β-carotene. Thirteen genes that encode enzymes in the carotenoid biosynthetic pathway were detected in papaya fruit transcriptome: two phytoene synthase (PSY1, PSY2), two phytoene desaturase (PDS1, PDS2), one ζ-carotene desaturase (ZDS), four lycopene cyclase (CYCB, LCYB1, LCYB2, LCYE), one β-carotene hydroxylase (CHYB), one carotene ε-monooxygenase (LUT1), one violaxanthin de-epoxidase (VDE), and one zeaxanthin epoxidase (ZEP). The results of RNA-Seq and RT-qPCR showed the expression of carotenoid biosynthetic pathway genes was consistent with the change of carotenoid content. Carotenoid biosynthetic pathways in the yellow peel and red flesh of papaya were analysed based on the major carotenoids accumulation and the expression patterns of key carotenoid biosynthesis pathway genes. There was only a β-branch of carotenoid biosynthesis in the flesh of papaya, while there were both α- and β-branch of carotenoid biosynthesis in papaya peel. In the process of papaya fruit ripening, the α-branch was inhibited and the β-branch was enhanced in the peel. The differential carotenoid accumulation and biosynthesis pathway genes expression in peel and flesh, lay a foundation for further study and provide further insights to control fruit color and improve fruit quality and appearance.

Background Since papaya is a typical climacteric fruit, exogenous ethylene (ETH) applications can induce premature and quicker ripening, while 1-methylcyclopropene (1-MCP) slows down the ripening processes. Differential gene expression in ETH or 1-MCP-treated papaya fruits accounts for the ripening processes. To isolate the key ripening-related genes and better understand fruit ripening mechanisms, transcriptomes of ETH or 1-MCP-treated, and non-treated (Control Group, CG) papaya fruits were sequenced using Illumina Hiseq2500. Results A total of 18,648 (1-MCP), 19,093 (CG), and 15,321 (ETH) genes were detected, with the genes detected in the ETH-treatment being the least. This suggests that ETH may inhibit the expression of some genes. Based on the differential gene expression (DGE) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, 53 fruit ripening-related genes were selected: 20 cell wall-related genes, 18 chlorophyll and carotenoid metabolism-related genes, four proteinases and their inhibitors, six plant hormone signal transduction pathway genes, four transcription factors, and one senescence-associated gene. Reverse transcription quantitative PCR (RT-qPCR) analyses confirmed the results of RNA-seq and verified that the expression pattern of six genes is consistent with the fruit senescence process. Based on the expression profiling of genes in carbohydrate metabolic process, chlorophyll metabolism pathway, and carotenoid metabolism pathway, the mechanism of pulp softening and coloration of papaya was deduced and discussed. We illustrate that papaya fruit softening is a complex process with significant cell wall hydrolases, such as pectinases, cellulases, and hemicellulases involved in the process. Exogenous ethylene accelerates the coloration of papaya changing from green to yellow. This is likely due to the inhibition of chlorophyll biosynthesis and the α-branch of carotenoid metabolism. Chy-b may play an important role in the yellow color of papaya fruit. Conclusions Comparing the differential gene expression in ETH/1-MCP-treated papaya using RNA-seq is a sound approach to isolate ripening-related genes. The results of this study can improve our understanding of papaya fruit ripening molecular mechanism and reveal candidate fruit ripening-related genes for further research.

以荔枝古树＂宋荔＂胚性愈伤组织为材料,采用接头染色体步移法,分离获得LcCu/Zn-SOD3启动子片段长度为1 426bp,命名为ProLcCSD3（GenBank登录号：KF672186.1）。生物信息学分析表明,该启动子含有多个逆境应答元件、激素应答元件、胚乳特异表达元件,且可能受WRKY和MYB等转录因子调控。通过双酶切方法,以ProLcCSD3替换载体pCAMBIA1301上的CaMv35S启动子,构建了重组质粒p1301-proLcCSD3-GUS,并成功转化农杆菌EHA105和GV3101。注射烟草的瞬时表达分析表明,该启动子片段可以驱动下游报告基因表达。转化拟南芥分析表明,该启动子驱动的下游GUS可以在拟南芥的根、茎、叶中表达,且可响应NaCl、PEG-6000、ABA、MeJA和损伤等非生物胁迫。研究表明,荔枝古树LcCu/Zn-SOD3可能参与多种非生物胁迫应答和激素信号转导途径。

MEE70/MSI1是拟南芥母性效应胚胎滞育基因,在植物胚胎发生过程中具有重要作用.采用RACE技术获得龙眼MEE70-1a(登录号KC492117)及其可变剪接体MEE70-1b(登录号KC492118)cDNA序列,克隆MSI1gDNA(登录号KC492126)序列.生物信息学分析预测DlMEE70-1a和DlMEE70-1b均为亲水不稳定的酸性蛋白,主要定位于过氧化物酶体和细胞核,各含有4和1个WD-repeat结构域.MEE70-1a和MEE70-1b在体细胞胚胎发生过程实时荧光定量PCR分析结果表明,DlMEE70-1a和DlMEE70-1b表达趋势以心形胚为界,除了DlMEE70-1b的不完全胚型紧实结构(ICpEC)到球形胚(GE)阶段之间,都表现出先降后升的趋势;胚性愈伤组织(EC)时期两者表达量一致且最高;心形胚(HE)时期,两者表达量一致且最低,另DlMEE70-1b在不完全胚性紧实结构(ICpEC)表达量与心形胚基本一致.这2个表达剂量是胚胎发生的重要保障,心形胚(HE)后,DlMEE70-1a被再次激活转录促进胚胎正常发育.从DlMEE70-1a和DlMEE70-1b的理化性质、WD重复结构域数量、亚细胞定位以及表达量和趋势,可以推测DlMEE70-1a需要DlMEE70-1b的协同来调控龙眼胚胎发育.

MEE70／MS11是拟南芥母性效应胚胎滞育基因，在植物胚胎发生过程中具有重要作用。采用RACE技术获得龙眼MEE70-1a（登录号KC492117）及其可变剪接体MEE70-1b（登录号KC492118）eDNA序列，克隆MSII gDNA（登录号KC492126）序列。生物信息学分析预测D1MEE70-1a和D1MEE70-1b均为亲水不稳定的酸性蛋白．主要定位于过氧化物酶体和细胞核．各含有4和1个WD—repeat结构域。MEE70-1a和MEE70-1b在体细胞胚胎发生过程实时荧光定量PCR分析结果表明．DIMEE70-1a和DlMEE70-1b表达趋势以心形胚为界．除了DIMEE70-1b的不完全胚型紧实结构（ICpEC）到球形胚fGE）阶段之间，都表现出先降后升的趋势；胚性愈伤组织（EC）时期两者表达量一致且最高：心形胚fHE）时期，两者表达量一致且最低，另DIMEE70-1b在不完全胚性紧实结构（ICpEC）表达量与心形胚基本一致。这2个表达剂量是胚胎发生的重要保障，心形胚（HE）后，DIMEE70-1a被再次激活转录促进胚胎正常发育。从D1MEE70-1a和D1MEE70-1b的理化性质、WD重复结构域数量、亚细胞定位以及表达量和趋势．可以推测DIMEE70-1a需要DIMEE70-1b的协同来调控龙眼胚胎发育。

Thirty-eight samples of wild banana leaves from Rixi town of Fuzhou were collected as materials, the technical system of ISSR marker was used, and their genetic structures were analyzed by related software STRUCTURE and NTSYS in the study. The results showed that based on 13 primers, 116 stable DNA fragments were obtaimed, of which polymorphic band number was 70, accounting for 60.34%,which revealed a relative high level of genetic diversity. The results of NTSYS and STRUCTURE analysis showed that 38 wild materials could be divided into 6 groups. The Q-value analysis showed that there was a certain exchang of genetic information in the natural populations of wild banana in Rixi town, Fuzhou city.

Two different mRNA transcripts and their genomic sequence of Fe-Superoxide dismutase gene were obtained with homologous and RACE from embryogenic callus of an ancient litchi tree ‘ Songli',and the bioinformatics were analyzed to illustrate its role in the process of in vitro reservation.The full length of LcFeSOD cDNA was about 1 285 bp,containing a 753-nucleotides-long open reading frame(ORF) which encoding a protein of 250 amino acid residues,named as LcFe-SOD7a.Bioinformatics analysis showed that the protein encoded by LcFe-SOD7a with transmembrane helices was hydrophilic,stable and acidic protein,mainly located in the microbody(peroxisome) and cytoplasm,and had a variety of phosphorylation sites.The genomic sequence of LcFe-SOD7a with a length of 2 284 bp was consisted of seven introns,with the fourth intron 920 bp,the longest.Meanwhile,an intron kept alternative splicing gene,named LcFe-SOD7b,which encoding a protein of 207 amino acid residues was obtained,which causing protein encoding terminated in advance.

In order to explore molecular mechanism of fruit ripening and the key softening-related genes,cDNA-AFLP technique was used to identify differentially expressed genes during papaya fruit ripening.Fifty transcript-derived fragments(TDFs)were obtained from the analysis of semi-yellow and color-break stage of papaya fruit.Bioinformatics analysis showed that 28 TDFs were homologous to the known functional genes,which 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.Using specific primers designed from the sequences of TDFs,four differential expression genes were cloned by rapid amplification of cDNA ends(RACE).Semi-quantitative RT-PCR analysis showed that the expression of four genes changed accordingly during fruit ripening,indicating the possible role of these genes in the ripening of papaya.

The full-length GMP cDNA was cloned by RACE-PCR from papaya and the gene w as designated as CpGMP(GenBank accession No.FJ489652).Open reading frame which encoded 361 amino acids was cloned from cDNA.DNA sequence including 4 extrons and 3 introns was cloned from genomic DNA.Sequencing analysis showed that the amino acid sequence was 90.30%,89.47%,88.92%,8 8.09%,85.87% homologous with GMP from Malpighia glabra,peach, tomato,Arabidopsis thaliana and rice,respectively.Semi-quantitative RTPCR analysis showed that the expression of CpGMP increased gradually with pa paya fruit maturating and decreased when fruit began to soften.

Two full-length polygalacturonase inhibiting protein(PGIP) cDNA and DNA were cloned by RT-PCR from papaya and the genes were named as CpPGIP1 and CpPGIP2 respectively.The full length CpPGIP1 was 984 bp which encoding a protein with 325 amino acids.The full length CpPGIP2 was 1 025 bp which coding for 326 amino acids.Sequencing analysis showed that the identical amino acid sequence of the two genes was 63.53%,while the identical nucleotide sequence was 66.54%,and no intron sequences were found in the two genes.The amino acid sequences of the two proteins both contained plant PGIP specific conserved leucine repeat sequence LXXLXXLXXLXLXXNXLXGXIPXX.The gene expression patterns in papaya fruit at different ripening stages were analyzed by semi-quantitative RT-PCR.The results showed that the expression patterns of the two genes basically showed similar trend.The expression of CpPGIP was the highest in green stage and decreased gradually with papaya fruit maturing.

High quality RNA was extracted from papaya pulp.Then the double-stranded cDNA was obtained through Super SMARTTM PCR cDNA Synthesis Kit.A full-length cDNA clone encoding Actin (Genbank accession number: FJ696416) was amplified by RACE-PCR from papaya.The full length cDNA of CpActin was 1 533 bp with an ORF of 1 134 bp encoding a protein with 377 amino acids.Sequence analysis showed that the amino acid sequence was 98.94%,98.67%,96.29%,94.69% identical with poplar,grape,rice and Arabidopsis thaliana respectively.Phylogenic tree was constructed based on the amino acid sequences of Actin from partial higher species,mammals and fungi using NeighborJoining method.The Actin from papaya and grape had the shortest evolutionary distance.Semi-Quantitative RT-PCR analysis showed that the expression of CpActin at different ripening stages had no notable difference.CpActin could be used as internal control gene in papaya gene expression analysis.

The rapid softening of fruit during postharvest storage has led to the limited shelf life of papaya. Transformation methods of liquid-shaking and soaking, mediated by Agrobacterium tumefaciens harboring an RNAi-β-Gal two-T-DNA plant expression vector p2301/TTRG, were compared and optimized for the efficiency in co-transformation of embryogenic calli of papaya. The suitable kanamycin concentration for the selection of transformants was 150 mg·L-1 based on the antibiotic sensitivity measurements. Under this concentration, the transgenic plantlets were obtained by both the two methods, but the transformation efficiency was low. The results of orthogonal experiments indicated that AS concentration was most important among the factors which influenced the transformation efficiency in soaking method. The co-cultivation time, bacterial culture OD and infection time functioned equally. The calculated best combination consisted of 100 μmol·L-1 AS, bacterial culture OD 0.2, 20 min for infection and 3 d for co-cultivation. Two of the five regenerated plantlets were proved to be cotransformed by PCR assay and southern blot, and one of them was induced to root and transplanted. The study will facilitate the breeding of transgenic papaya lines, which are marker-free and softening-resistant.

Rapid softening of papaya fruit(Carica papaya L.)during postharvest storage is closely related to the expression of β-Gal gene.By using conventional and inverse PCR,a 4501 bp genomic sequence coding for papaya β-Gal gene was isolated,containing 17 introns and exons with one base different from the cDNA sequence.Bioinformatic analysis of this gene revealed that this protein belonged to the member of family 35 of glycoside hydrolyase superfamily 42,and had a closer genetic relationship with Arabidopsis thaliana,but farther with Persea americana and Picea sitchensis.Additionally,the predicted protein included a signal peptide located extracellularly,indicating again the possible involvement of this enzyme in the degradation of cell wall matrix and fruit softening.The corresponding promoter of β-Gal was also isolated,and proved to be of expression specificity in the fruit.CaMV 35S promoter on the vector p2301/TTRG was replaced by β-Gal promoter,leading to the construction of β-Gal-RNAi Two-T-DNA plant expression vector p2301/BPTTRG.The transformation of p2301/BPTTRG into Agrobactrium tumefaciens was confirmed by restriction enzyme analysis and PCR assay.

Complete 3′ and partial 5′ terminal sequence of pectate lyase gene were obtained by rapid amplification of cDNA ends(RACE) from papaya fruit.Using two specific primers,open reading frame encoding 385 amino acids was cloned from genome DNA.Sequencing analysis showed that the amino acid sequence was 83.38%,75.76%,73.68% and 65.96% identical with that of the strawberry,peach,mango and arabidopsis thaliana,respectively.The cDNA of pectate lyase conserved domain was inserted into between the CaMV 35S promoter and Nos terminator of the expression vector pBI121 with reverse orientation.So,antisense expression vector pBP was obtained and transformed into agrobacterium tumefaciens EHA105.

The conserved region of β-Gal gene of papaya (Carica papaya L.) pulp was cloned,which encoded a key enzyme of β-galactosidase involved in the cell wall degradation. The RNAi intermediate expression vector of pKAN/RG was constructed containing β-Gal gene in an inverted repeat orientation with the help of pKANNIBAL vector. hptⅡ gene of the modified pCAMBIA 1300 vector was replaced by the hairpin structure of pKAN/RG,which resulted in the formation of intermediate expression vector of p1300-/MFRG. Single T-DNA region of p1300-/MFRG was isolated and incorporated into the pCAMBIA 2301 vector to produce the RNAi Two-T-DNA plant expression vector of p2301/TTRG. The transformation of p2301/TTRG into Agrobacterium tumefaciens EHA 105 was confirmed by restriction enzyme analysis and PCR assay. The embryogenic calli of papaya which had kanamycin resistance and GUS expression were obtained by genetic transformation.

On the basis of one cDNA-AFLP expressed sequence tag(EST) from longan(Dimocarpus longan Lour.) cotyledon embryos which is 84%homologous to Antirrhinum majus,the full length cDNA of glyceraldehyde-3-phosphate dehydrogenase(GAPDH) gene was cloned by rapid-amplification of cDNA ends(RACE).The gene consisted of 1395 bp encoding a protein of 336 amino acids,with 71 bp and 316 bp at 5′-UTR and 3′-UTR,respectively.The amino acid sequence comparison with the GAPDH gene of Oryza sativa,Vitis vinifera,Musa acuminata,Arabidopsis thaliana,and Ginkgo biloba showed that identity was all higher than 85%.The sequence was accepted and released by GenBank(accession number: FJ694011).

The finger map of longan (Dimdcarpus longan Lour. 'Honghezi') cotyledon embryo RNA was established by cDNA-AFLP,which extracted from thirty-five-day (early) and fifty-day (late) cotyledon embryos after anthesis,respectively. There were 41 differential fragments cloned and sequence analysed,in which 21 differential expression fragments had high homology with some known genes,the function mainly involved in the abaxial polarity of lateral organ,glycolysis,energy metabolism,ion transport,cell wall elongation,cell cycle regulation,RNA transcription,RNA translation and regulation,protein phosphorylation regulation and protein degradation,signal transduction and so on. While the others had low or no homology with known genes.

Three methods for the extraction of genomic DNA from leaves of tissue culture seedlings of Carica papaya L.were compared.For the samples ground in the ice bath,genomic DNA with high quality was extracted by the simple processes of modified CTAB method,which could be applied to PCR assay of transgenic papaya,restriction enzyme digestion,and the subsequent inverse PCR.SDS-KAc method was unable to get rid of RNA.DNA extracted by kit showed low production and quality.