Abstract
【Objective】The aim of this study was to clone full-length cDNA of phenylalanine ammonia-lyase(PAL) gene(CP-PAL) in seed coat of the hulled Cucurbita pepo, analyze its expressions in seed coat development of the hulled and hull-less C. pepo, thus providing a theoretical support for revealing the mechanism of seed development and function of accumulation of lignin in seed coat development. 【Method】 The full sequence of CP-PAL was cloned by RT-PCR and RACE techniques. The bioinformatics method was used to analyze cDNA sequence and deduced amino acid sequence, and the real-time PCR and 2-△△Ct method were used to analyze the expression profile of PAL gene in the whole period of seed coat development. 【Result】 The full-length sequence of CP-PAL consists of 1 720 bp with an intact open reading frame of 1 359 bp, 5′UTR of 114 bp, 3′UTR of 236 bp, polyA of 11 bp, encoding a polypeptide of 452 amino acids. The molecular weight of CP-PAL was 48.86 kD, pI was 6.5, total number of atoms was 6 885 and the formula was C2158H3449N607O657S14. Homology analysis showed that the CP-PAL nucleotide sequences and deduced amino acids were highly homologous to that of PAL gene in Cucumis sativus. CP-PAL contained three functional domains of PAL-HAL, PLN02457, phe_am_lyase and the typical PAL enzyme active site sequence(GTITASGDLVPLSYIA), and it was one member of Lyase_I_Like superfamily. CP-PAL was most likely to be located in the cytoplasm and endoplasmic reticulum, without signal peptide and leader peptide. And it was non-transmembrane and soluble protein. Besides, CP-PAL included casein kinase II phosphorylation site for four, protein kinase C phosphorylation site for six, N-myristoylation site for twelve and N-glycosylation site for two. In addition, CP-PAL had eighteen serine phosphorylation sites, six threonine phosphorylation sites and five tyrosine phosphorylation sites. Random coil was the maximum structural part in the protein secondary structure of CP-PAL, alpha helix and extended strand dispersed in whole protein, N-terminal region was presented mainly in the form of random coil, and C-terminal domain was existed in the form of extended strand. The multiple sequence alignment based on the deduced amino acid sequences of CP-PAL and other PALs of 14 plants showed the functional region was conserved, differences in the N-end. Phylogenetic tree analysis indicated that CP-PAL was very closely related to PAL of C. sativus. The main structural element in CP-PAL protein tertiary structure was α-Helix, less in β-Turn and random coil. Real-time PCR analysis revealed that PAL gene in hull-less C. pepo showed an opposite tendency to hulled C. pepo. The expression of PAL gene in hulled C. pepo was increased, but the expression in hull-less C. pepo was decreased after 20d of self-pollination. The expression of PAL gene in hull-less C. pepo was lower than that in hulled C. pepo. 【Conclusion】The gene CP-PAL which related to lignin synthesis was firstly cloned and characterized in seed coat of hulled C. pepo. The results of study indicated that it may affect seed coat development of the naked kernel variety from C. pepo by regulating the synthesis of lignin.
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Claims 10
Claim · model-written summary
The full-length CP-PAL sequence is 1 720 bp with an open reading frame of 1 359 bp.
- Taxon
- Cucurbita pepo
Cucurbita pepot0 - Gene
- CP-PALe1
- Outcome
- full-length sequencee1
- Value
- 1 720 bpe1
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“Cucurbita pepo”
e1“The full-length sequence of CP-PAL consists of 1 720 bp with an intact open reading frame of 1 359 bp”
Claim · model-written summary
CP-PAL encodes a polypeptide of 452 amino acids.
- Taxon
- Cucurbita pepoCucurbita pepot0
- Outcome
- polypeptidee2
- Value
- 452 amino acidse2
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“Cucurbita pepo”
e2“encoding a polypeptide of 452 amino acids”
Claim · model-written summary
CP-PAL nucleotide and deduced amino acid sequences are highly homologous to PAL of Cucumis sativus.
- Taxon
- Cucurbita pepoCucurbita pepot0
- Gene
- CP-PALe3
- Outcome
- homologye3
- Comparator
- PAL gene in Cucumis sativuse3
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“Cucurbita pepo”
e3“Homology analysis showed that the CP-PAL nucleotide sequences and deduced amino acids were highly homologous to that of PAL gene in Cucumis sativus”
Claim · model-written summary
CP-PAL contains three functional domains and the typical PAL active site sequence.
- Taxon
- Cucurbita pepoCucurbita pepot0
- Gene
- CP-PALe4
- Outcome
- functional domainse4
- Value
- threee4
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“Cucurbita pepo”
e4“CP-PAL contained three functional domains of PAL-HAL, PLN02457, phe_am_lyase and the typical PAL enzyme active site sequence(GTITASGDLVPLSYIA)”
Claim · model-written summary
CP-PAL is most likely located in the cytoplasm and endoplasmic reticulum and has no signal peptide.
- Taxon
- Cucurbita pepoCucurbita pepot0
- Gene
- CP-PALe5
- Outcome
- most likely to be located in the cytoplasm and endoplasmic reticulume5
- Qualifier
- without signal peptide and leader peptidee5
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“Cucurbita pepo”
e5“CP-PAL was most likely to be located in the cytoplasm and endoplasmic reticulum, without signal peptide and leader peptide”
Claim · model-written summary
CP-PAL is very closely related to PAL of Cucumis sativus in phylogeny.
- Taxon
- Cucurbita pepoCucurbita pepot0
- Gene
- CP-PALe6
- Outcome
- very closely relatede6
- Comparator
- PAL of C. sativuse6
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“Cucurbita pepo”
e6“Phylogenetic tree analysis indicated that CP-PAL was very closely related to PAL of C. sativus”
Claim · model-written summary
PAL expression increased in hulled C. pepo after 20 days of self-pollination.
- Taxon
- Cucurbita pepoCucurbita pepot0
- Material
- hulled C. pepoe7
- Treatment
- self-pollinatione7
- Gene
- PALe7
- Outcome
- expression of PAL genee7
- Direction
- increasede7
- Qualifier
- 20de7
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“Cucurbita pepo”
e7“The expression of PAL gene in hulled C. pepo was increased, but the expression in hull-less C. pepo was decreased after 20d of self-pollination”
Claim · model-written summary
PAL expression decreased in hull-less C. pepo after 20 days of self-pollination.
- Taxon
- Cucurbita pepoCucurbita pepot0
- Material
- hull-less C. pepoe7
- Treatment
- self-pollinatione7
- Gene
- PALe7
- Outcome
- expression of PAL genee7
- Direction
- decreasede7
- Qualifier
- 20de7
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“Cucurbita pepo”
e7“The expression of PAL gene in hulled C. pepo was increased, but the expression in hull-less C. pepo was decreased after 20d of self-pollination”
Claim · model-written summary
PAL expression was lower in hull-less than in hulled C. pepo.
- Taxon
- Cucurbita pepoCucurbita pepot0
- Material
- hull-less C. pepoe0
- Gene
- PALe0
- Outcome
- expression of PAL genee0
- Direction
- lowere0
- Comparator
- hulled C. pepoe0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“Cucurbita pepo”
e0“The expression of PAL gene in hull-less C. pepo was lower than that in hulled C. pepo”
Claim · model-written summary
CP-PAL may affect seed coat development of the naked kernel variety by regulating lignin synthesis.
- Taxon
- Cucurbita pepoCucurbita pepot0
- Material
- naked kernel varietye0
- Gene
- CP-PALe1
- Compound
- lignine0
- Outcome
- seed coat developmente0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“Cucurbita pepo”
e0“The results of study indicated that it may affect seed coat development of the naked kernel variety from C. pepo by regulating the synthesis of lignin”
e1“The gene CP-PAL which related to lignin synthesis was firstly cloned and characterized in seed coat of hulled C. pepo”