非洲菊GjPAL的克隆及表达分析

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

摘要/Abstract

摘要：

为了解非洲菊PAL基因编码蛋白的理化性质和表达模式,以非洲菊品种“玲珑”为材料,利用RACE和RT-PCR技术克隆获得4个PAL基因(命名为GjPAL1-GjPAL4),利用生物信息学方法研究它们的结构及其编码蛋白的特性。利用qPCR检测GjPALs在不同组织部位和不同逆境胁迫处理下的表达模式。序列分析显示:4个GjPAL的CDS为2 115-2 136 bp,编码蛋白长度为704-711 aa,均无信号肽的稳定酸性亲水蛋白。实时荧光定量PCR结果显示,GjPAL1在叶中表达量最高,GjPAL2-GjPAL4均在根中表达量最高;4个GjPALs的表达量均受低温显著抑制;GjPAL1和GjPAL2的表达受SA、NaCl和PEG显著抑制;GjPAL3和GjPAL4在SA处理下呈先升后降的表达趋势,GjPAL3的表达受NaCl和PEG的显著诱导;隐地疫霉处理条件下,GjPAL1和GjPAL4的表达量显著升高,GjPAL2的表达量显著降低,而GjPAL3的表达量呈先降后升趋势。GjPALs广泛参与非洲菊对不同逆境的应答过程,各成员在不同逆境处理下的作用存在一定差异。

关键词: 非洲菊, 苯丙氨酸解氨酶, 基因克隆, 生物信息学分析, 表达模式

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

郝向阳, 刘范, 武欢, 王斌, 孙雪丽, 项蕾蕾, 王天池, 赖钟雄, 程春振. 非洲菊GjPAL的克隆及表达分析[J]. 生物技术通报, 2021, 37(6): 13-23.

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.

图/表 9

表1 基因克隆和定量引物信息

Table 1 Information of the primers used for gene cloning and qRT-PCR

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

图1 PCR产物电泳图 1-7分别为:GjPAL1 3'RACE、GjPAL3 RT-PCR、GjPAL1 5'RACE、GjPAL1 RT-PCR、GjPAL4 3'RACE、GjPAL2 RT-PCR和GjPAL4 RT-PCR验证结果;M:DL5000 DNA marker(条带从下到上依次为:100 bp、250 bp、500 bp、750 bp、1 000 bp、1 500 bp、2 000 bp、3 000 bp和5 000 bp);m:DL2000 DNA marker(条带从下到上依次为:100 bp、250 bp、500 bp、750 bp、1 000 bp和2 000 bp)

Fig. 1 Electrophoresis result of PCR products 1-7 is the electrophoresis result of GjPAL1 3' RACE, GjPAL3 RT-PCR, GjPAL1 5' RACE, GjPAL1 RT-PCR, GjPAL4 3' RACE, GjPAL2 RT-PCR and GjPAL4 RT-PCR products, respectively. M: DL5000 DNA marker (the band from bottom to top is 100 bp, 250 bp, 500 bp, 750 bp, 1 000 bp, 1 500 bp, 2 000 bp, 3 000 bp and 5 000 bp, respectively). m: DL2000 DNA marker (the band from bottom to top is 100 bp, 250 bp, 500 bp, 750 bp, 1000 bp and 2000 bp, respectively)

表2 4个GjPALs蛋白基本理化性质分析结果

Table 2 Physicochemical properties of the four GjPALs

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

图2 GjPAL1-GjPAL4(A-D)蛋白保守结构域和motif(E)分析

Fig. 2 Conserved domains (A-D) and motifs (E) of GjPAL1-4

图3 不同植物PAL氨基酸序列的多重序列比对黑框代表酶活性中心序列,红框代表典型的保守区域

Fig. 3 Multiple alignment result of PALs from different plant species Characters in the black box represent the enzyme activity center and the red box represent the typical conserved region of the PAL enzyme activity center

图4 4个GjPALs三维结构的预测

Fig. 4 Predicted four-dimensional structures of the four GjPALs

表3 GjPAL跨膜结构域及磷酸化修饰位点分析结果

Table 3 Result of transmembrane structure and phospho-rylation sites in GjPALs

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

图5 GjPALs和其他植物PALs系统发育树 I:双子叶植物;II:单子叶植物;III:裸子植物;IV:蕨类植物

Fig. 5 Phylogenetic tree for GjPALs and PALs from some other plant species I: Dicotyledons. II: Monocotyledons. III: Gymnosperm. IV: Pteridophyte

图6 GjPAL基因在不同组织部位和不同逆境处理下的表达情况 A:不同组织器官;B:SA处理;C:NaCl处理;D:PEG处理;E:低温处理;F:隐地疫霉接种;*表示0.05水平上显著差异;**表示0.01水平上极显著差异

Fig.6 Relative expression of GjPAL genes in different tissues and organs and under different stress treatments A: Different organs. B: SA treatment. C: NaCl treatment. D: PEG treatment. E: Cold stress treatment. F: Phytophthora cryptogea innoculation treatment. “*” indicates significant difference at the 0.05 level, and “**” indicates very significant difference at the 0.01 level

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