|
|
|
| Cloning of Promoter of Legumin Gene Mt1g072600 from Medicago truncatula and Organs Expression Analysis |
| WEI Qi-Chao*, WANG Ya-Li, ZHOU Yan |
| School of Life Science and Technology, Henan Institute of Science and Technology, Xinxiang 453003, China |
|
|
|
|
Abstract In the practice of genetic engineering, such as improving the nutritional quality of seeds and using seed-based bioreactor to produce proteins with industrial or medicinal value, it is necessary to use seed specific promoters to drive the transcription of exogenous genes. Seed storage proteins are the general name of a large number of storage proteins synthesized and stored in the process of seed ripening in higher plants, the promoters of the genes coding seed storage proteins serve as an important source for seed specific promoters. In order to obtain the seed specific promoter of Medicago truncatula, 2 075 bp promoter sequence (named PMt1g072600) of upstream of coding region of the major storage protein legumin gene Mt1g072600 was cloned, and the putative cis-acting elements of the promoter were predicted using workflow embed in PLACE and PlantCARE, the results showed that this sequence contained E-box, A/T rich element, P-box and other cis-acting elements with highly relevant features to seed specific promoter. The plant expression vector to drive the GUS gene using this promoter was constructed and transformed into Arabidopsis thaliana, the result of analysis of GUS expression level in transgenic A. thaliana seeds showed that the expression of GUS driven by PMt1g072600 was significantly higher than that drived by PCaMV35S. The results of histochemical staining of tissues/organs of transgenic A. thaliana showed that GUS driven by PMt1g072600 showed the specificity of seed expression.This study identified a seed specific promoter PMt1g072600 of M. truncatula, which can be used to drive the specific transcription of exogenous genes in transgenic plant seeds. This study also provides a reference for selection and utilization of seed specific promoters in plant genetic engineering.
|
|
Received: 08 December 2020
|
|
|
|
Corresponding Authors:
* wei_qc@aliyun.com
|
|
|
|
[1] 韩宝达, 李立新. 2010. 植物种子贮藏蛋白质及其细胞内转运与加工[J]. 植物学报, 45(4): 492-505.
(Han B D, Li L X.2010. Seed storage proteins and their intracellular transport and processing[J]. Chinese Bulletin of Botany, 45(4): 492-505.)
[2] 刘峰, 汪小东, 赵彦鹏, 等. 2014. 棉花种子特异表达的LEA启动子克隆及功能验证[J]. 棉花学报, 26(4): 290-294.
(Liu F, Wang X D, Zhao Y P, et al.2014. Isolation and functional characterization of the seed-specific promoter of LEA gene from cotton (Gossypium hirsutum L.)[J]. Cotton Science, 26(4): 290-294.)
[3] 吉仁花, 张文波, 林晓飞, 等. 2020. 杂交构树UDP-葡萄糖脱氢酶基因编码蛋白的亚细胞定位及其启动子5'端缺失片段的功能分析[J]. 植物研究, 40(6): 932-942.
(Ji R H, Zhang W B, Lin X F, et al.2020. Subcellular localization of the protein coded by the UDP-glucose dehydrogenase gene from Paper mulberry and functional of its promoter 5'-end deletion fragment[J]. Bulletin of Botanical Research, 40(6): 932-942.)
[4] 石磊, 齐飞艳, 苗利娟, 等. 2018. 一个花生早期胚特异性表达基因AhDGAT3启动子的克隆及功能分析[J]. 中国油料作物学报, 40(1): 25-34.
(Shi L, Qi F Y, Miao L J, et al.2018. Cloning and functional analysis of a novel early embryo-specific expressed AhDGAT3 promoter from Arachis hypogaea L.[J]. Chinese Journal of Oil Crop Sciences, 40(1): 25-34.)
[5] 司爱君, 杨维才, 谢宗铭, 等. 2018. 陆地棉叶片特异性表达启动子的克隆与表达分析[J]. 西南农业学报, 31(4): 646-652.
(Si A J, Yang W C, Xie Z M, et al.2018. Cloning and expression analysis of cotton leaf-specific promoter[J]. Southwest China Journal of Agricultural Sciences, 31(4): 646-652.)
[6] 孙全喜, 徐洪明, 李春娟, 等. 2020. 花生种子特异启动子AHSSP1的克隆及功能分析[J]. 核农学报, 34(3): 460-467.
(Sun Q X, Xu H M, Li C J, et al.2020. Isolation and functional analysis of seed specific promoter AHSSP1 from peanut (Arachis hypogaea L.)[J]. Journal of Nuclear Agricultural Sciences, 34(3): 460-467.)
[7] 唐克轩, 沈乾, 付雪晴, 等. 2014. 植物次生代谢产物生物反应器研究进展[J]. 中国农业科技导报, 16(1): 7-15.
(Tang K X, Shen Q, Fu X Q, et al.2014. Research progress on plant secondary metabolite bioreactor[J]. Journal of Agricultural Science and Technology, 16(1): 7-15.)
[8] 王海兰, 贾庆利, 赵翠珠, 等. 2018. 甘蓝型油菜种子特异表达油体蛋白启动子PBnOA03的克隆及功能分析[J]. 西北植物学报, 27(8): 1152-1160.
(Wang H L, Jia Q L, Zhao C Z, et al.2018. Cloning and function analysis of oleosin promoter PBnOA03 from Brassica napus[J]. Acta Agriculturae Boreali-occidentalis Sinica, 27(8): 1152-1160.)
[9] 杨晶, 杜林娜, 王法微, 等. 2018. 新型植物生物反应器研究进展[J]. 生物产业技术, (5): 104-109.
(Yang J, Du L N, Wang F W, et al. 2018. Research on novel plant bioreactors[J]. Biotechnology & Business, (5): 104-109.)
[10] 尹梦回, 董静, 李先碧, 等. 2008. 烟草绒毡层特异启动子pTA29在棉花中的表达特性[J]. 作物学报, 34(12): 2092-2098.
(Yin M H, Dong J, Li X B, et al.2008. Expression characteristics of tobacco tapetum-specific promoter pTA29 in cotton[J]. Acta Agronomica Sinica, 34(12): 2092-2098.)
[11] 朱飞雪, 杜建材, 王照兰, 等. 2007. 五种不同苜蓿的种子蛋白指纹图谱研究[J]. 中国草地学报, 29(5): 1-7.
(Zhu F X, Du J C, Wang Z L, et al.2007. A study on the seed protein fingerprints of five alfalfa species[J]. Chinese Journal of Grassland, 29(5): 1-7.)
[12] Benedito V A, Torres-Jerez I, Murray J D, et al.2008. A gene expression atlas of the model legume Medicago truncatula[J]. The Plant Journal, 55(3): 504-513.
[13] Boothe J, Nykiforuk C, Shen Y, et al.2010. Seed-based expression systems for plant molecular farming[J]. Plant Biotechnology Journal, 8(5): 588-606.
[14] Branca A, Paape T D, Zhou P, et al.2011. Whole-genome nucleotide diversity, recombination, and linkage disequilibrium in the model legume Medicago truncatula[J]. Proceedings of the National Academy of Sciences of the USA, 108(42): E864-E870.
[15] Bustos M M, Guiltinan M J, Jordano J, et al.1989. Regulation of β-glucuronidase expression in transgenic tobacco plants by an A/T-rich, cis-acting sequence found upstream of a French bean β-phaseolin gene[J]. The Plant Cell, 1(9): 839-853.
[16] Chandrasekharan M B, Bishop K J, Hall T C.2003. Module-specific regulation of the β-phaseolin promoter during embryogenesis[J]. The Plant Journal, 33(5): 853-866.
[17] Che Z, Cao X, Chen G, et al.2020. An effective combination of codon optimization, gene dosage, and process optimization for high-level production of fibrinolytic enzyme in Komagataella phaffii (Pichia pastoris)[J]. BMC Biotechnology, 20(1): 1-13.
[18] Chen H, Zeng Y, Yang Y, et al.2020. Allele-aware chromosome-level genome assembly and efficient transgene-free genome editing for the autotetraploid cultivated alfalfa[J]. Nature Communications, 11(1): 1-11.
[19] Clough S J, Bent A F.1998. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana[J]. The Plant Journal, 16(6): 735-743.
[20] Ezcurra I, Wycliffe P, Nehlin L, et al.2000. Transactivation of the Brassica napus napin promoter by ABI3 requires interaction of the conserved B2 and B3 domains of ABI3 with different cis-elements: B2 mediates activation through an ABRE, whereas B3 interacts with an RY/G-box[J]. The Plant Journal, 24(1): 57-66.
[21] Fujiwara T, Beachy R N.1994. Tissue-specific and temporal regulation of a β-conglycinin gene: Roles of the RY repeat and other cis-acting elements[J]. Plant Molecular Biology, 24(2): 261-272.
[22] He J, Benedito V A, Wang M, et al.The Medicago truncatula gene expression atlas web server[J]. BMC Bioinformatics, 2009, 10(1): 1-9.
[23] He Y, Ning T, Xie T, et al.2011. Large-scale production of functional human serum albumin from transgenic rice seeds[J]. Proceedings of the National Academy of Sciences of the USA, 108(47): 19078-19083.
[24] Higo K, Ugawa Y, Iwamoto M, et al.1999. Plant cis-acting regulatory DNA elements (PLACE) database: 1999[J]. Nucleic Acids Research, 27(1): 297-300.
[25] Hood E E, Bailey M R, Beifuss K, et al.2003. Criteria for high-level expression of a fungal laccase gene in transgenic maize[J]. Plant Biotechnology Journal, 1(2): 129-140.
[26] Howe K L, Contreras-Moreira B, De Silva N, et al.2020. Ensembl genomes 2020-enabling non-vertebrate genomic research[J]. Nucleic Acids Research, 48(D1): D689-D695.
[27] Jefferson R A, Kavanagh T A, Bevan M W.1987. GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants[J]. The EMBO Journal, 6(13): 3901-3907.
[28] Koltunow A M, Truettner J, Cox K H, et al.1990. Different temporal and spatial gene expression patterns occur during anther development[J]. The Plant Cell, 2(12): 1201-1224.
[29] Laloum T, De Mita S, Gamas P, et al.2013. CCAAT-box binding transcription factors in plants: Y so many?[J]. Trends in Plant Science, 18(3): 157-166.
[30] Le Signor C, Gallardo K, Prosperi J M, et al.2005. Genetic diversity for seed protein composition in Medicago truncat-ula[J]. Plant Genetic Resources: Characterization and utilization, 3(1): 59-71.
[31] Lessard P A, Allen R D, Fujiwara T, et al.1993. Upstream regulatory sequences from two β-conglycinin genes[J]. Plant Molecular Biology, 22(5): 873-885.
[32] Liu S J, Yue Q J, Zhang W.2015. Structural and functional analysis of an asymmetric bidirectional promoter in Arabidopsis thaliana[J]. Journal of Integrative Plant Biology, 57(2): 162-170.
[33] Mariani C, Beuckeleer M D, Truettner J, et al.1990. Induction of male sterility in plants by a chimaeric ribonuclease gene[J]. Nature, 347(6295): 737-741.
[34] Nykiforuk C L, Boothe J G, Murray E W, et al.2006. Transgenic expression and recovery of biologically active recombinant human insulin from Arabidopsis thaliana seeds[J]. Plant Biotechnology Journal, 4(1): 77-85.
[35] Qu L Q, Takaiwa F.2004. Evaluation of tissue specificity and expression strength of rice seed component gene promoters in transgenic rice[J]. Plant Biotechnology Journal, 2(2): 113-125.
[36] Ramessar K, Capell T, Christou P.2008. Molecular pharming in cereal crops[J]. Phytochemistry Reviews, 7(3): 579-592.
[37] Shen C, Du H, Chen Z, et al.2020. The Chromosome-level genome sequence of the autotetraploid alfalfa and resequencing of core germplasms provide genomic resources for alfalfa research[J]. Molecular Plant, 13(9): 1250-1261.
[38] Stuart D A, Nelsen J.1988. Isolation and characterization of alfalfa 7S and 11S seed storage protein[J]. Journal of plant physiology, 132(2): 129-133.
[39] Sunilkumar G, Connell J P, Smith C W, et al.2002. Cotton α-globulin promoter: Isolation and functional characterization in transgenic cotton, Arabidopsis, and tobacco[J]. Transgenic Research, 11(4): 347-359.
[40] Tiwari S, Mishra D K, Roy S, et al.2009. High level expression of a functionally active cholera toxin B: Rabies glycoprotein fusion protein in tobacco seeds[J]. Plant Cell Reports, 28(12): 1827-1836.
[41] Ueda T, Wang Z, Pham N, et al.1994. Identification of a transcriptional activator-binding element in the 27-kilodalton zein promoter, the -300 element[J]. Molecular and Cellular Biology, 14(7): 4350-4359.
[42] Wu X L, Li B Z, Zhang W Z, et al.2017. Genome-wide landscape of position effects on heterogeneous gene expression in Saccharomyces cerevisiae[J]. Biotechnology for Biofuels, 10(1): 1-10.
[43] Young N D, Debellé F, Oldroyd G E, et al.2011. The Medicago genome provides insight into the evolution of rhizobial symbioses[J]. Nature, 480(7378): 520-524. |
|
|
|
