Cloning of Cassava Phenylalanine Ammonia Lyase Genes and Their Responses to Low Temperature Stress

doi:10.3969/j.issn.1000-2561.2019.03.010

Abstract

Abstract:

Cassava phenylalanine ammonia-lyase encoding genes (MePALs) were identified from the whole genome sequence data. Six MePALs genes, MePAL1-6, were subsequently cloned by RT-PCR from the leaves of the cultivar KU50 and their expression patterns were analyzed, together with PAL activity, total phenolic content, flavonoid content and total antioxidant capacity, in the leaves under low temperature stress treatment (7 ℃). It was found that low temperature treatment resulted in rapid leaf wilting and curling and correspondingly a significant increase in PAL activity, total phenol content, flavonoid content and total antioxidant capacity in the leaves. Real-time PCR analysis showed that the relative expression level of MePAL1 and MePAL2 was 0.83 and 1.19 respectively before low temperature treatment, which was significantly higher than the other 4 MePALs. Low temperature treatment increased the expression of all the six MePAL genes and the up-regulation of MePAL5 was the highest (increased from 50.5 to 142.5 folds). Our preliminary results showed that low temperature treatment increased the expression of MePALs, promoted the synthesis of total phenols and flavonoids, and enhanced anti-oxidative capacities in cassava leaves.

Key words: cassava, low temperature stress, phenylalanine ammonia-lyase gene, flavonoids, antioxidant

WU Yuanhang,LIU Qin,LIU Pandao,GUO Pengfei,LI Min,JIANG Lingyan,LUO Lijuan. Cloning of Cassava Phenylalanine Ammonia Lyase Genes and Their Responses to Low Temperature Stress[J]. Chinese Journal of Tropical Crops, 2019, 40(3): 483-489.

Figures/Tables 7

References 28

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引物名称 Primer name	正向引物序列 Forward sequence ( 5'-3' )	反向引物序列 Reverse sequence ( 5'-3' )
MePAL1全长引物	ATGGAAGTGTCCGGCGGAA	CTAACAGATTGGCAGAGGTGTG
MePAL2全长引物	ATGGACTTCTGTCACGACAA	TTAGGAGATGGGAAGAGGAGC
MePAL3全长引物	ATGGCAACAATCTCTCAGAATGG	TTAACATATTGGAAGAGGGGCAC
MePAL4全长引物	ATGGCAACAGTCTCTCAGAAT	TTAACAGATTGGAAGAGGGGCA
MePAL5全长引物	ATGGAGTTTTGTGAGGCTCACAAT	TTAGCAGATTGGAAGAGGAGCA
MePAL6全长引物	ATGGCTGCAAATGGCGTTG	TTAAAACAGAGGAGCTCTCTCTTTC
MePAL1-定量引物	ATCCTTTGCAGAAGCCTAAGCAG	TAATGCCAATCTAGCGTTGTCC
MePAL2-定量引物	AGCCTAGACAAGATCGTTATGCTC	TTGTGCAAACATGAGTTTCCC
MePAL3-定量引物	CTTATCAGCTATTTTCGCCGAAG	TCAATCTGAGGACCTAGCCAT
MePAL4-定量引物	GCACACAGTGAAGAACACCGTA	TAAGGCATGATCCACAAGCAC
MePAL5-定量引物	GCTCCAATTTCTTGCCAATCCTG	GCCGTTGATGCCCATTGTCA
MePAL6-定量引物	TGAATATCTCTCGTGGGACT	AGAATCTCCCACCGAATACCAG
ef1α-内参引物	TCCACTGGGTAGATTCGCTGTTA	TAGCTCCACTTGGGTCCTTCTT

引物名称 Primer name	正向引物序列 Forward sequence ( 5'-3' )	反向引物序列 Reverse sequence ( 5'-3' )
MePAL1全长引物	ATGGAAGTGTCCGGCGGAA	CTAACAGATTGGCAGAGGTGTG
MePAL2全长引物	ATGGACTTCTGTCACGACAA	TTAGGAGATGGGAAGAGGAGC
MePAL3全长引物	ATGGCAACAATCTCTCAGAATGG	TTAACATATTGGAAGAGGGGCAC
MePAL4全长引物	ATGGCAACAGTCTCTCAGAAT	TTAACAGATTGGAAGAGGGGCA
MePAL5全长引物	ATGGAGTTTTGTGAGGCTCACAAT	TTAGCAGATTGGAAGAGGAGCA
MePAL6全长引物	ATGGCTGCAAATGGCGTTG	TTAAAACAGAGGAGCTCTCTCTTTC
MePAL1-定量引物	ATCCTTTGCAGAAGCCTAAGCAG	TAATGCCAATCTAGCGTTGTCC
MePAL2-定量引物	AGCCTAGACAAGATCGTTATGCTC	TTGTGCAAACATGAGTTTCCC
MePAL3-定量引物	CTTATCAGCTATTTTCGCCGAAG	TCAATCTGAGGACCTAGCCAT
MePAL4-定量引物	GCACACAGTGAAGAACACCGTA	TAAGGCATGATCCACAAGCAC
MePAL5-定量引物	GCTCCAATTTCTTGCCAATCCTG	GCCGTTGATGCCCATTGTCA
MePAL6-定量引物	TGAATATCTCTCGTGGGACT	AGAATCTCCCACCGAATACCAG
ef1α-内参引物	TCCACTGGGTAGATTCGCTGTTA	TAGCTCCACTTGGGTCCTTCTT

基因名称 Gene name	基因ID Gene ID	编码区长度 CDS/bp	蛋白 Protein
基因名称 Gene name	基因ID Gene ID	编码区长度 CDS/bp	长度 Length/aa	分子量 MW/ku	等电点 pI
MePAL1	Manes.04G018000	2148	715	77.77	6.06
MePAL2	Manes.07G098700	2112	703	76.97	6.00
MePAL3	Manes.08G008400	2139	712	77.43	6.31
MePAL4	Manes.09G063700	2151	716	77.71	5.90
MePAL5	Manes.10G047500	2133	710	77.59	6.03
MePAL6	Manes.16G098200	2136	711	78.16	5.98

基因名称 Gene name	基因ID Gene ID	编码区长度 CDS/bp	蛋白 Protein
基因名称 Gene name	基因ID Gene ID	编码区长度 CDS/bp	长度 Length/aa	分子量 MW/ku	等电点 pI
MePAL1	Manes.04G018000	2148	715	77.77	6.06
MePAL2	Manes.07G098700	2112	703	76.97	6.00
MePAL3	Manes.08G008400	2139	712	77.43	6.31
MePAL4	Manes.09G063700	2151	716	77.71	5.90
MePAL5	Manes.10G047500	2133	710	77.59	6.03
MePAL6	Manes.16G098200	2136	711	78.16	5.98