Cloning and Expression Analysis of GjPAL Genes in Gerbera jamesonni

doi:10.13560/j.cnki.biotech.bull.1985.2020-1343

Abstract

Abstract:

To clarify the physicochemical properties of the proteins encoded by the gerbera PAL genes,and to explore their expression pattern, four plenylalanine ammonia lyase genes(named as GjPAL1-GjPAL4)were successfully cloned from Gerbera jamesonni cv ‘Linglong’ using RACE and RT-PCR techniques. Then,a series of bioinformatics software were used to study their sequence structures and characteristics of their encoded proteins. qPCR was used to show their expression patterns in different tissues and organs,and under different stress treatments. The results from sequence analysis showed that the coding sequence(CDS)length of 4 GjPALs ranged from 2 115 to 2 136 bp,which encoded protein containing 704-711 amino acids,respectively. All the encoded proteins of the 4 GjPALs were found to be stable acidic hydrophilic proteins without signal peptide. qRT-PCR results demonstrated that GjPAL1 expressed the highest in leaf,while GjPAL2-GjPAL4 expressed the highest in root. Under low temperature treatment,the expressions of the 4 GjPALs were all significantly inhibited. GjPAL1 and GjPAL2 were significantly inhibited by SA,NaCl and PEG treatments,GjPAL3 and GjPAL4 showed ‘rise-fall’ expression patterns under SA treatment,while GjPAL3 expression was found to be significantly induced by NaCl and PEG treatments. Under Phytophthora cryptogea inoculation treatment,GjPAL1 and GjPAL4 were significantly induced,GjPAL2 was significantly suppressed,and GjPAL3 showed a ‘fall-rise’ expression pattern. Results indicated that GjPALs widely participated in the gerbera stress responses,but their response patterns to different stresses varied greatly.

Key words: Gerbera jamesonni, phenylalanine ammonia-lyase, gene cloning, bioinformatics analysis, expression pattern

HAO Xiang-yang, LIU Fan, WU Huan, WANG Bin, SUN Xue-li, XIANG Lei-lei, WANG Tian-chi, LAI Zhong-xiong, CHENG Chun-zhen. Cloning and Expression Analysis of GjPAL Genes in Gerbera jamesonni[J]. Biotechnology Bulletin, 2021, 37(6): 13-23.

Figures/Tables 9

References 36

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引物名称Primer name	引物序列Primer sequence(5'-3')	产物大小Product size/bp	用途Application
GjPAL1F-outer	GGATTACGGGTTCAAAGGT		3' RACE
GjPAL1F-inner	GCTCCAGTTTCTTGCTAATCC
GjPAL4F-outer	TAAGCCAAGTAGCCAAGAAGGT
GjPAL4F-inner	AGGACTTGCTTCGTGTGGTT
GjPAL1R-outer	CGGCGTTCAAGAATCTGA		5'RACE
GjPAL1R-inner	GTGACACCGTAACTATCAGTCCC		5'RACE
GjPAL1F	GAGTCCAGTGTGTGAAAC	2 301	RT-PCR
GjPAL1R	AATGGTGAGCCTCTTCCGA	2 301
GjPAL2F	TGGATCATACCAATGGAAATGAC	2 137
GjPAL2R	CACTTATGAAATGGGAAGAGGG	2 137
GjPAL3F	ATGGACAGTAAAGCCGTCAAGATC	2 204
GjPAL3R	CTAACAAATTGGAAGAGGAACAC	2 204
GjPAL4F	GAGTCTAGTGTGTGAAACTTTGATAGC	2 117
GjPAL4R	TTAACATATCGGAAGAGGCTCAC	2 117
GjPAL1-qF	TTCCGAACAGAATCAAGGC	125	qRT-PCR
GjPAL1-qR	TGACCCTTACACATTGCCG	125
GjPAL2-qF	TGCATGAATAGTGATCCATTG	145
GjPAL2-qR	GATTCTGACAACTCCACCTGT	145
GjPAL3-qF	TCACATAACCAGCCATTTCG	173
GjPAL3-qR	ACCATCCGCTTCACTTCGT	173
GjPAL4-qF	CGATAATCAATGGAGAACGG	172
GjPAL4-qR	TCCACCATCTGTTTCACCTC	172
18sF	TCAAAGCAAGCCTACGCTCT	125
18sR	GCTTTCGCAGTTGTTCGTCT	125

引物名称Primer name	引物序列Primer sequence(5'-3')	产物大小Product size/bp	用途Application
GjPAL1F-outer	GGATTACGGGTTCAAAGGT		3' RACE
GjPAL1F-inner	GCTCCAGTTTCTTGCTAATCC
GjPAL4F-outer	TAAGCCAAGTAGCCAAGAAGGT
GjPAL4F-inner	AGGACTTGCTTCGTGTGGTT
GjPAL1R-outer	CGGCGTTCAAGAATCTGA		5'RACE
GjPAL1R-inner	GTGACACCGTAACTATCAGTCCC		5'RACE
GjPAL1F	GAGTCCAGTGTGTGAAAC	2 301	RT-PCR
GjPAL1R	AATGGTGAGCCTCTTCCGA	2 301
GjPAL2F	TGGATCATACCAATGGAAATGAC	2 137
GjPAL2R	CACTTATGAAATGGGAAGAGGG	2 137
GjPAL3F	ATGGACAGTAAAGCCGTCAAGATC	2 204
GjPAL3R	CTAACAAATTGGAAGAGGAACAC	2 204
GjPAL4F	GAGTCTAGTGTGTGAAACTTTGATAGC	2 117
GjPAL4R	TTAACATATCGGAAGAGGCTCAC	2 117
GjPAL1-qF	TTCCGAACAGAATCAAGGC	125	qRT-PCR
GjPAL1-qR	TGACCCTTACACATTGCCG	125
GjPAL2-qF	TGCATGAATAGTGATCCATTG	145
GjPAL2-qR	GATTCTGACAACTCCACCTGT	145
GjPAL3-qF	TCACATAACCAGCCATTTCG	173
GjPAL3-qR	ACCATCCGCTTCACTTCGT	173
GjPAL4-qF	CGATAATCAATGGAGAACGG	172
GjPAL4-qR	TCCACCATCTGTTTCACCTC	172
18sF	TCAAAGCAAGCCTACGCTCT	125
18sR	GCTTTCGCAGTTGTTCGTCT	125

蛋白 Protein	分子量 Molecular weight/kD	分子式 Formula	等电点pI	脂肪系数 Aliphatic index	不稳定系数 Instability index	总亲水性 GRAVY
GjPAL1	77 052.27	C₃₄₀₅H₅₄₅₂N₉₃₈O₁₀₃₃S₃₁	6.18	90.49	31.79	-0.134
GjPAL2	76 538.09	C₃₃₈₄H₅₃₈₉N₉₂₅O₁₀₄₄S₂₅	5.53	91.97	34.20	-0.125
GjPAL3	77 326.50	C₃₄₂₈H₅₄₇₀N₉₄₀O₁₀₃₆S₂₈	5.93	92.07	34.24	-0.091
GjPAL4	77 091.13	C₃₄₀₆H₅₄₅₃N₉₄₁O₁₀₃₈S₂₈	6.18	91.05	32.98	-0.152

蛋白 Protein	分子量 Molecular weight/kD	分子式 Formula	等电点pI	脂肪系数 Aliphatic index	不稳定系数 Instability index	总亲水性 GRAVY
GjPAL1	77 052.27	C₃₄₀₅H₅₄₅₂N₉₃₈O₁₀₃₃S₃₁	6.18	90.49	31.79	-0.134
GjPAL2	76 538.09	C₃₃₈₄H₅₃₈₉N₉₂₅O₁₀₄₄S₂₅	5.53	91.97	34.20	-0.125
GjPAL3	77 326.50	C₃₄₂₈H₅₄₇₀N₉₄₀O₁₀₃₆S₂₈	5.93	92.07	34.24	-0.091
GjPAL4	77 091.13	C₃₄₀₆H₅₄₅₃N₉₄₁O₁₀₃₈S₂₈	6.18	91.05	32.98	-0.152

蛋白 Protein	长度 Length/aa	卷曲螺旋结构 Coiled coil structure	跨膜结构域Transmembrane domain		磷酸化修饰位点Phosphorylation site
蛋白 Protein	长度 Length/aa	卷曲螺旋结构 Coiled coil structure	外→内Outside→Inside	内→外Inside→Outside	S	T	Y
GjPAL1	708	无	3	5	33	21	7
GjPAL2	704	有	3	7	34	20	7
GjPAL3	711	有	5	6	30	24	8
GjPAL4	708	无	2	5	36	19	8