Cloning And Expression Analysis Of Genes And MYB Transcription Factors Involved In Lignin Biosynthesis Pathway In Pomegranate (Punica Granatum L.)

Pomegranate(Punica granatum L.)plant was a multifunction species with a wide range of economic value,such as fruit for eating,flower for ornamental,tree for virescence and exocarp for Chinese medicines.Pomegranate seeds consist of three parts,the aril,seed coat and cotyledons.Pomegranate cultivar with hard-seeded fruit has higher lignin content in seed coat and is inconvenient to eat,while that with soft-seeded fruit is a precious resource which has lower lignin content in seed coat and can be eaten with the aril.Currently,there is a little research on the mechanism of development and formation of soft-seeded fruit.Three cultivars of pomegranate,‘Tunisiruanzi',‘Huiliruanzi' and ‘Hongyushizi' with different seed hardness were used as materials in this study to investigate the effect of relevant genes and MYB transcription factors on lignin biosynthesis and formation of soft-seeded fruit.Four genes involved in lignin biosynthesis pathway and two MYB transcription factors were cloned from cultivar ‘Hongyushizi' by rapid amplification of cDNA ends(RACE).Expression patterns of those genes were analyzed by Real time-PCR in different organs,seed coat of different developmental stages and different cultivars.The main results were obtained as follows:1 The partial 3'-end sequence of PgPAL and the full-length cDNA of PgCCR,PgCOMT,PgLAC were cloned by RACE.Bioinformatics analysis showed that PgPAL had high identity with Ricinus communis,Vitis vinifera,Populus trichocarpa as 90%.The cDNA of PgCCR(GenBank accession No.: KM881713)was 1,269 bp and encoded a protein of 338 amino acid residues,the identity of PgCCR with Hevea brasiliensis,Pyrus pyrifolia,Hibiscus cannabinus,Eucalyptus gunnii and Populus trichocarpa were over 82%.The cDNA of PgCOMT(GenBank accession No.:KJ713968)was 1,456 bp and encoded a protein of 370 amino acid residues,the identity of PgCOMT with Clarkia breweri,Ricinus communis,Prunus dulcis and Eucalyptus camaldulensis were over 86%.The cDNA of PgLAC(GenBank accession No.: KM881711)was 2,128 bp and encoded a protein of 571 amino acid residues,the identity of PgLAC with Vitis vinifera,Nicotiana tomentosiformis,Theobroma cacao and Malus domestica were over 80%.2 Two MYB transcription factors PgMYB and PgMYB1 were cloned from cultivar‘Hongyushizi' by RACE.Bioinformatics analysis showed that both of the two geneswere typical R2R3-MYB transcription factors in plant with two MYB DNA binding domains at their N-terminus.The cDNA of PgMYB(GenBank accession No.:KM014568)was 1,088 bp and encoded a protein of 306 amino acid residues,the identity of PgMYB with Leucaena leucocephala,Populus and Arabidopsis thaliana were over 84%.The cDNA of PgMYB1(GenBank accession No.: KM881712)was987 bp and encoded a protein of 263 amino acid residues,the identity of PgMYB1 with Antirrhinum majus,Theobroma cacao,Eucalyptus grandis and Nicotiana tomentosiformis were over 71%.3 Real time-PCR analysis revealed that all of the genes were expressed in pomegranate organs leaf,petal and stem.The relative expression levels of PgCCR,PgCOMT,PgLAC and PgMYB were all highest in stem and lowest in leaf,except for the genes PgPAL and PgMYB1 which were expressed highly in petal and lowly in leaf and stem.4 In pomegranate seed coat of different developmental stages,the expression of PgPAL,PgCCR and PgLAC basically presented one trend and reached the peak at 80 day after full bloom.The expression of PgCOMT declined at first,and then rose at the last stage.The MYB transcription factors PgMYB1 and PgMYB expressed in a similar trend which were highest at the first stage and then gradually declined along with the ascension in development processes and total lignin content.5 In seed coat of pomegranate cultivars with different seed hardness,the expression pattern of PgCCR,PgCOMT,PgLAC and PgMYB1 were basically the same,and had a similar trend with seed hardness and total lignin content.The genes expression in ‘Tunisiruanzi' and ‘Huiliruanzi' were lower than that in ‘Hongyushizi'.While PgPAL and PgMYB expressed relatively higher in ‘Tunisiruanzi' and‘Huiliruanzi' than that in ‘Hongyushizi'.