家榆UpNOA1的克隆及其互作蛋白的筛选

doi:10.13560/j.cnki.biotech.bull.1985.2024-1143

摘要/Abstract

摘要：

目的一氧化氮相关蛋白NOA1与植物体内NO水平有关，可能参与植物多种生理活动。克隆UpNOA1，分析其在家榆各组织中的表达情况，筛选与UpNOA1互作的蛋白，为研究家榆UpNOA1的功能提供依据。方法以家榆（Ulmus pumila L.）为试验材料，克隆UpNOA1基因，运用生物信息学分析其结构域、进化树等，采用RT-qPCR分析UpNOA1的组织特异性表达情况。构建载体进行原核表达分析，获得纯化UpNOA1蛋白。利用His-PULL DOWN筛选并初步分析UpNOA1的互作蛋白。结果家榆UpNOA1编码区全长1 698 bp，共编码565个氨基酸，包含1个保守的GTP/Mg²⁺结合位点。经预测，UpNOA1蛋白定位于叶绿体。UpNOA1蛋白与美国榆UaNOA1蛋白序列同源性最高。RT-qPCR分析发现，UpNOA1在叶片中表达量最高，种子次之。经His-PULL DOWN筛选，共获得131个UpNOA1互作蛋白，且与遗传信息处理相关的核糖体蛋白最多。经比对，有25个蛋白与抗逆和GTP相关。结论克隆获得UpNOA1，在叶片中表达量最高，种子次之。鉴定到131个UpNOA1互作蛋白，其中有25个蛋白与抗逆和GTP相关。

关键词: 家榆, NOA1, 组织特异性表达, PULL DOWN, 互作蛋白

Abstract:

Objective Nitric oxide-related protein NOA1 is associated to the levels of NO in plants, which may be involved in various physiological activities of plants. The UpNOA1 gene was cloned, its tissue-specific expression in elm (Ulmus pumila L.) was analyzed, and the proteins interacting with UpNOA1 was screened, which may provide the basis for studying the function of UpNOA1 in elm. Method Ulmus pumila L. was used as the experimental material, and the NO-related UpNOA1 gene was cloned. The domain architecture and phylogenetic relationships were analyzed using bioinformatics approaches. Tissue-specific expression of UpNOA1 was assessed via RT-qPCR. A vector was constructed for prokaryotic expression analysis, and the resulting UpNOA1 protein was purified. Finally, the UpNOA1 interaction proteins were screened and analyzed by His-PULL DOWN. Result The UpNOA1 gene possesses a 1 698 bp open reading frame, encoding 565 amino acids and containing a conserved GTP/Mg²⁺ binding sites. Subcellular localization prediction using Plant-mPLoc indicated that the UpNOA1 protein is targeted to chloroplasts. Phylogenetic analysis revealed that the UpNOA1 protein shows the highest sequence homology to UaNOA1 protein from American elm (Ulmus americana) and clusters with it in the phylogenetic tree. RT-qPCR analysis revealed that the expression of UpNOA1 gene is the highest in the leaves, followed by the seeds. A total of 131 proteins were identified as UpNOA1-interacting proteins via His-PULL DOWN assay. KEGG and GO enrichment analyses indicated a predominance of ribosomal proteins is associated with genetic information processing among the interacting proteins. Following annotation and comparison, a list of 25 candidate proteins potentially involved in stress resistance and GTP binding was compiled. Conclusion UpNOA1 gene is cloned. The highest expression is found in leaves, followed by elm seeds. A total of 131 UpNOA1 interacting proteins are identified, of which 25 are associated with stress resistance and GTP binding.

Key words: Ulmus pumila L., NOA1, tissue specific expression, PULL DOWN, interaction protein

刘晓天, 何毓琦, 刘畅, 薛华. 家榆UpNOA1的克隆及其互作蛋白的筛选[J]. 生物技术通报, 2025, 41(6): 297-306.

LIU Xiao-tian, HE Yu-qi, LIU Chang, XUE Hua. Cloning of UpNOA1 Gene from Ulmus pumila L. and Screening of Its Interacting Proteins[J]. Biotechnology Bulletin, 2025, 41(6): 297-306.

图/表 9

表1 引物信息

Table 1 Primer information

引物Primer	序列Sequence （5′‒3′）
UpNOA1 PCR F	ATGGCGTCCAAAACCCTCTC
UpNOA1 PCR R	TCAAAAATACCATCTTGGTCTTGTT
UpNOA1 pMAL F	TCCATGGGCGATATCGGATCCATGGCGTCCAAAACCCTCTCAAC
UpNOA1 pMAL R	TTAATTACCTGCAGGGAATTCGTGATGGTGATGGTGATGGTGATGAAAATACCATCTT
qUpActin F	TATAATGAATTGCGTGTAGCCC
qUpActin R	CATAGCAGGAGTGTTGAAGGTC
qUpNOA1 F	CATCTTGAAGGCTCTACCGG
qUpNOA1 R	TTGTAACCCTTTCCACTCCTG

图1 UpNOA1蛋白的理化性质及结构分析A：UpNOA1蛋白保守结构域分析；B：UpNOA1蛋白二级结构分析（蓝色线条：α-螺旋；紫色线条：延伸链；绿色线条：β-转角；黄色线条：无规卷曲）；C：UpNOA1蛋白三级结构（a）及活性中心（b）预测

Fig. 1 Physicochemical properties and structural analysis of UpNOA1 proteinA: Analysis of conserved domains of UpNOA1 protein. B: Secondary structure analysis of UpNOA1 protein (Blue line: α‍-helix. Purple line: Extended strand. Green line: β-sheet. Yellow line: Random coil). C: Prediction of tertiary structure (a) and active center (b) of UpNOA1 protein

图2 家榆与其他物种NOA1氨基酸的序列比对红线标出氨基酸为GTP结合位点。At：拟南芥；Nb：本氏烟草；Zj：枣；Pa：杏；Pd：扁桃；Ra：黑莓；Cs：大麻；Rc：月季；Mn：桑；Ua：美国榆

Fig. 2 Amino acid sequence alignment of NOA1 from Ulmus pumila and other speciesThe red lines indicate amino acids as GTP-binding sites. At: Arabidopsis thaliana; Nb: Nicotiana benthamiana; Zj: Ziziphus jujuba; Pa: Prunus armeniaca; Pd: Prunus dulcis; Ra: Rubus argutus; Cs: Cannabis sativa; Rc: Rosa chinensis; Mn: Morus notabilis; Ua: Ulmus americana

图3 家榆与其他物种NOA1的系统进化树

Fig. 3 Phylogenetic tree of NOA1 from U. pumila and other species

图4 UpNOA1在家榆不同组织中的表达分析** P<0.01

Fig. 4 Gene expressions of UpNOA1 in different tissues of U. pumila

图5 UpNOA1的克隆与原核表达A：UpNOA1的PCR产物电泳图（M：Marker DL2000）；B：UpNOA1-His蛋白纯化SDS-PAGE分析图（1：阴性对照；2：原核表达UpNOA1-His蛋白；3：纯化后的UpNOA1-His蛋白）；C：家榆UpNOA1的核苷酸及氨基酸序列；D：pMAL-c5x-UpNOA1菌落PCR产物电泳图（M：Marker DL2000；1-10：pMAL-c5x-UpNOA1菌落PCR产物）

Fig. 5 Cloning and prokaryotic expression of UpNOA1A: Electrophoresis analysis of UpNOA1 PCR products (M: Marker DL2000). B: Purification and SDS-PAGE analysis of UpNOA1-His protein (1: negative control; 2: protein sample from prokaryotic expression of UpNOA1-His; 3: the purified UpNOA1-His protein). C: Nucleotide sequence and amino acid sequence of UpNOA1 in U. pumila. D: Electrophoresis analysis of pMAL-c5x-UpNOA1 PCR products from bacterial colony (M: Marker DL2000. 1-10: PCR identification of different colonies of pMAL-c5x-UpNOA1)

图6 His-PULL DOWN筛选互作蛋白SDS-PAGE分析图M：Marker；1：原核表达蛋白；2：原核表达蛋白过Ni-NTA柱流穿液；3：家榆种子总蛋白过UpNOA1-Ni-NTA柱流穿液；4：纯化的UpNOA1诱饵蛋白与其互作蛋白洗脱液；5：家榆种子总蛋白。箭头指示位置为诱饵蛋白条带，红框内为下拉蛋白条带

Fig. 6 SDS-PAGE analysis of interacting proteins screened by His-PULL DOWNM: Marker; 1: protein sample from prokaryotic expression; 2: the prokaryotic expressed protein in lane 1 flowed through the Ni-NTA Agarose; 3: the seed proteins flow through the UpNOA1-Ni-NTA Agarose; 4: purified UpNOA1 bait protein and its interacting protein elutes; 5: total seed protein of U. pumila. The arrow indicates the location of the bait protein strip, and the red box shows the PULL-DOWN protein strip

图7 UpNOA1互作蛋白GO通路（A）及KEGG通路（B）分析

Fig. 7 GO (A) and KEGG (B) pathway analysis of UpNOA1 interacting proteins

表2 UpNOA1互作候选蛋白

Table 2 Candidates of UpNOA1 interacting proteins

序号 Serial number	蛋白名称 Protein name	功能注释 Function annotation	分子量 Molecular weight （kD）	等电点 Isoelectric point	蛋白特征性肽段数量 Number of characteristic peptides
1	TRINITY_DN4364_c1_g2	预测：热休克70 kD蛋白，线粒体［葡萄（Vitis vinifera）］	30.1	9.47	1
2	TRINITY_DN3442_c0_g1	18.1 kD I类热休克蛋白样蛋白［开心果（Pistacia vera）］	17.9	5.77	1
3	TRINITY_DN3511_c0_g1	16.9 kD 热休克蛋白［白桦（Betula platyphylla）］	17.5	5.71	4
4	TRINITY_DN4927_c0_g1	11S球蛋白前体同工型1A ［爱玉子（Ficus pumila var. awkeotsang）］	63.2	7.02	24
5	TRINITY_DN1797_c0_g1	11-S种子储藏蛋白［安德森寄生木（Parasponia andersonii）］	67.4	6.83	25
6	TRINITY_DN7461_c0_g1	类似球蛋白的种子储藏蛋白 At2g28490 ［显著桑（Morus notabilis）］	63.1	6.01	14
7	TRINITY_DN14490_c0_g1	晚期胚胎发生丰富蛋白，第3类［显著桑（Morus notabilis）］	14.1	4.92	5
8	TRINITY_DN17317_c0_g1	2-Cys过氧化物还原酶BAS1，叶绿体［普通山羊草（Aegilops tauschii subsp. tauschii）］	26.9	6.35	1
9	TRINITY_DN28842_c0_g1	甘油醛-3-磷酸脱氢酶，胞质型［向日葵（Helianthus annuus）］	21.7	5.95	2
10	TRINITY_DN1098_c0_g5	超氧化物歧化酶，部分序列［欧洲山毛榉（Fagus sylvatica）］	18.5	7.09	2
11	TRINITY_DN4794_c0_g1	种子生物素结合蛋白SBP65类似蛋白［石榴（Punica granatum）］	34.1	6.61	1
12	TRINITY_DN6469_c0_g1	抗渗蛋白类似蛋白［加州栎（Quercus lobata）］	27.0	7.61	4
13	TRINITY_DN5650_c0_g3	假定蛋白 EUGRSUZ_I01938 ［巨桉（Eucalyptus grandis）］	21.5	9.72	3
14	TRINITY_DN17167_c0_g1	光合作用NDH复合体类囊体腔定位亚基5 （PNSL5），叶绿体［向日葵（Helianthus annuus）］	20.5	8.68	1
15	TRINITY_DN12443_c0_g1	未注释蛋白 LOC115700795 同工型 X2 ［大麻（Cannabis sativa）］	19.9	7.88	5
16	TRINITY_DN10189_c0_g1	富甘氨酸蛋白2类似蛋白［枣（Ziziphus jujuba）］	18.9	6.74	2
17	TRINITY_DN18240_c0_g1	富甘氨酸RNA结合蛋白［番茄（Solanum lycopersicum）］	17.3	5.71	1
18	TRINITY_DN4384_c0_g1	预测：有丝分裂检查点蛋白BUB3.3类似蛋白［欧洲野草莓（Fragaria vesca subsp. vesca）］	17.4	5.99	5
19	TRINITY_DN175_c4_g1	预测：UPF0098蛋白 CPn_0877/CP_0992/CPj0877/CpB0906 ［梅花（Prunus mume）］	18.4	5.59	5
20	TRINITY_DN43339_c0_g1	假定蛋白 EW026_g2737 ［Phlebia centrifuga］	24.0	6.05	1
21	TRINITY_DN12101_c0_g2	GTP结合蛋白SAR1A类似蛋白［Rhodamnia argentea］	22.1	7.47	5
22	TRINITY_DN17498_c0_g5	推定小GTP酶超家族，Rab类型［牛樟（Cinnamomum micranthum f. kanehirae）］	11.7	9.41	1
23	TRINITY_DN6469_c0_g1	类渗透蛋白［加州栎（Quercus lobata）］	27.0	7.61	4
24	TRINITY_DN12488_c0_g1	生长素结合蛋白ABP19a类似蛋白［月季（Rosa chinensis）］	21.5	9.06	1
25	TRINITY_DN1250_c0_g1	推定4-羟基-4-甲基-2-氧代戊二酸醛缩酶2 ［桑树（Morus notabilis）］	17.7	5.99	3

表2 UpNOA1互作候选蛋白

Table 2 Candidates of UpNOA1 interacting proteins

序号 Serial number	蛋白名称 Protein name	功能注释 Function annotation	分子量 Molecular weight （kD）	等电点 Isoelectric point	蛋白特征性肽段数量 Number of characteristic peptides
1	TRINITY_DN4364_c1_g2	预测：热休克70 kD蛋白，线粒体［葡萄（Vitis vinifera）］	30.1	9.47	1
2	TRINITY_DN3442_c0_g1	18.1 kD I类热休克蛋白样蛋白［开心果（Pistacia vera）］	17.9	5.77	1
3	TRINITY_DN3511_c0_g1	16.9 kD 热休克蛋白［白桦（Betula platyphylla）］	17.5	5.71	4
4	TRINITY_DN4927_c0_g1	11S球蛋白前体同工型1A ［爱玉子（Ficus pumila var. awkeotsang）］	63.2	7.02	24
5	TRINITY_DN1797_c0_g1	11-S种子储藏蛋白［安德森寄生木（Parasponia andersonii）］	67.4	6.83	25
6	TRINITY_DN7461_c0_g1	类似球蛋白的种子储藏蛋白 At2g28490 ［显著桑（Morus notabilis）］	63.1	6.01	14
7	TRINITY_DN14490_c0_g1	晚期胚胎发生丰富蛋白，第3类［显著桑（Morus notabilis）］	14.1	4.92	5
8	TRINITY_DN17317_c0_g1	2-Cys过氧化物还原酶BAS1，叶绿体［普通山羊草（Aegilops tauschii subsp. tauschii）］	26.9	6.35	1
9	TRINITY_DN28842_c0_g1	甘油醛-3-磷酸脱氢酶，胞质型［向日葵（Helianthus annuus）］	21.7	5.95	2
10	TRINITY_DN1098_c0_g5	超氧化物歧化酶，部分序列［欧洲山毛榉（Fagus sylvatica）］	18.5	7.09	2
11	TRINITY_DN4794_c0_g1	种子生物素结合蛋白SBP65类似蛋白［石榴（Punica granatum）］	34.1	6.61	1
12	TRINITY_DN6469_c0_g1	抗渗蛋白类似蛋白［加州栎（Quercus lobata）］	27.0	7.61	4
13	TRINITY_DN5650_c0_g3	假定蛋白 EUGRSUZ_I01938 ［巨桉（Eucalyptus grandis）］	21.5	9.72	3
14	TRINITY_DN17167_c0_g1	光合作用NDH复合体类囊体腔定位亚基5 （PNSL5），叶绿体［向日葵（Helianthus annuus）］	20.5	8.68	1
15	TRINITY_DN12443_c0_g1	未注释蛋白 LOC115700795 同工型 X2 ［大麻（Cannabis sativa）］	19.9	7.88	5
16	TRINITY_DN10189_c0_g1	富甘氨酸蛋白2类似蛋白［枣（Ziziphus jujuba）］	18.9	6.74	2
17	TRINITY_DN18240_c0_g1	富甘氨酸RNA结合蛋白［番茄（Solanum lycopersicum）］	17.3	5.71	1
18	TRINITY_DN4384_c0_g1	预测：有丝分裂检查点蛋白BUB3.3类似蛋白［欧洲野草莓（Fragaria vesca subsp. vesca）］	17.4	5.99	5
19	TRINITY_DN175_c4_g1	预测：UPF0098蛋白 CPn_0877/CP_0992/CPj0877/CpB0906 ［梅花（Prunus mume）］	18.4	5.59	5
20	TRINITY_DN43339_c0_g1	假定蛋白 EW026_g2737 ［Phlebia centrifuga］	24.0	6.05	1
21	TRINITY_DN12101_c0_g2	GTP结合蛋白SAR1A类似蛋白［Rhodamnia argentea］	22.1	7.47	5
22	TRINITY_DN17498_c0_g5	推定小GTP酶超家族，Rab类型［牛樟（Cinnamomum micranthum f. kanehirae）］	11.7	9.41	1
23	TRINITY_DN6469_c0_g1	类渗透蛋白［加州栎（Quercus lobata）］	27.0	7.61	4
24	TRINITY_DN12488_c0_g1	生长素结合蛋白ABP19a类似蛋白［月季（Rosa chinensis）］	21.5	9.06	1
25	TRINITY_DN1250_c0_g1	推定4-羟基-4-甲基-2-氧代戊二酸醛缩酶2 ［桑树（Morus notabilis）］	17.7	5.99	3

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