Cloning of UpNOA1 Gene from Ulmus pumila L. and Screening of Its Interacting Proteins

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

Abstract

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

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.

Figures/Tables 9

References 41

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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

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

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

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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