Preliminary Studies on the Effect of GhCRE1 on Seed Germination in Gossypium hirsutum L.

doi:10.7668/hbnxb.20192894

Abstract

Abstract:

To clarify the function of the receptor gene for Gossypium hirsutum L. cytokinin receptor (GhCRE1) in the regulation of plant growth and development, and to provide material for studying the molecular mechanism of the GhCRE1 gene in regulating seed germination in Gossypium hirsutum, ultimately improving seed germination rates at the molecular level. The GhCRE1 gene was cloned and the fragment was ligated to the pCUbi1390 vector containing the Ubiquitin promoter using gene overexpression technology to construct GhCRE1 overexpression vector, while the sgRNA fragment of the Arabidopsis CRE1 gene was designed by CRISPR/Cas9 gene editing technology and the sgRNA nucleotide sequence was synthesized to construct the Arabidopsis CRE1 gene targeting knockout vector. The above two vectors were transformed into Agrobacterium and the inflorescence dip method was used to create GhCRE1 overexpression strains and gene editing loss-of-function mutants using Arabidopsis thaliana as the recipient material. Screening and identification by PCR and qPCR techniques, then, two strains of GhCRE1 Arabidopsis over-expressed and the CRE1-deficient Arabidopsis strains were obtained. After comparing the germination potential of harvested seeds with that of wild-type seeds on 1/2 MS medium, further clarify the function of GhCRE1 gene in affecting seed germination of Gossypium hirsutum. The results showed that the Arabidopsis GhCRE1 overexpression strains and CRISPR gene editing loss-of-function mutant strain were successfully obtained, after compiling the data related to seed germination potential, it was found that on the 2nd day, the germination potential of the two overexpression transgenic strains increased by about 15% and 11%, respectively, compared with the wild type, while the Arabidopsis CRE1 loss-of-function mutant germination potential decreased by about 15%. This study revealed that GhCRE1 gene overexpression significantly increased germination speed in Arabidopsis.

Key words: Gossypium hirsutum L., GhCRE1 gene, Overexpression, CRISPR/Cas9 technology, Seed germination

XU Minhui, Josee Ornella Musaniwabo, HAN Shaofan, LIU Yihan, DU Lihua, LIU Aiyu, WANG Qiming, TU Xiaoju. Preliminary Studies on the Effect of GhCRE1 on Seed Germination in Gossypium hirsutum L.[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(3): 19-26. doi: 10.7668/hbnxb.20192894.

/ / Recommend / Download Citations

URL: http://www.hbnxb.net/EN/10.7668/hbnxb.20192894

http://www.hbnxb.net/EN/Y2022/V37/I3/19

Figures/Tables 12

Fig.1 Schematic diagram of construction method of recombinant GhCRE1 overexpression vector

Tab.1 Primers used in screening and identification of Arabidopsis GhCRE1 overexpression plants

引物名称 Primer name	引物序列(5'-3') Primer sequence(5'-3')
Actin 11-F	CCACATGCTATTCTGCGTTTGGACC
Actin 11-R	CATCCCTTACGATTTCACGCTCTGC
GhCRE1-qF	CAGGCAGCAAAACGGACAC
GhCRE1-qR	CTCTTCCAGGCTGCCTCTTC

Tab.2 Primers used in construction of sgRNA expression cassette

引物名称 Primer name	引物序列(5'-3') Primer sequence(5'-3')
U-F	CTCCGTTTTACCTGTGGAATCG
gR-R	CGGAGGAAAATTCCATCCAC
CRE1-gRT1-F	CTGCTGCGTTTGAAAGAAAGTTTTAGAGCTAGAAAT
CRE1-U6T1-R	TTTCTTTCAAACGCAGCAGCAATCACTACTTCGTCT
lcyb-gRT1-F	ATGACGATGCTCGAGAGGCGTTTTAGAGCTAGAAAT
CRE1-U3T2-R	GTAATGAGTTGTGAGAGGATGACCAATGGTGCTTTG
CRE1-gRT2-F	TCCTCTCACAACTCATTACGTTTTAGAGCTAGAAAT
Bsa-C1-F	TTCAGAGGTCTCTCTCGACTAGTATGGAATCGGCAGCAAAGG
Bsa-C1-R	AGCGTGGGTCTCGTCAGGGTCCATCCACTCCAAGCTC
Bsa-C2-F	TTCAGAGGTCTCTCTGACACTGGAATCGGCAGCAAAGG
Bsa-GGR-R	AGCGTGGGTCTCGACCGACGCGTATCCATCCACTCCAAGCTC

Tab.3 Primers used in screening and identification of of CRE1 gene editing plants

引物名称 Primer name	引物序列(5'-3') Primer sequence(5'-3')
CRET1-F	GCTCTTTACAGGCGTGATTGT
CRET1-R	CCGCGCCAAATTATACGCTT
CRET2-F	AGAGCTAGTTTCGGAGACTTGTGAT
CRET2-R	GACTTCAAGAAACTATGGAGGTACT

Fig.2 Electrophoretic map of GhCRE1-CDS cloning M.DNA Marker DL1000;1,2. GhCRE1-CDS segment.

Fig.3 Electrophoresis test results of GhCRE1 recombinant overexpression vector construction A.PCR of recombinant vector colonies:M.DNA Marker DL2000;1-8.Colony PCR detection.B.Recombinant plasmid electrophoresis and digestion with Pst Ⅰ:M.DL10000 Marker;1.Recombinant plasmid digested with Pst Ⅰ;2.Recombinant plasmid.

Fig.4 Screening of GhCRE1 overexpression materials and expression analysis of GhCRE1 overexpression lines A.PCR assay of T₀ generation plants:M.DNA Marker DL2000;1-7.PCR electrophoresis bands for GhCRE1 overexpression detection.B.qPCR assay of GhCRE1 relative expression.

Fig.5 Electrophoretic detection results of sgRNA expression cassette construction of CRE1 gene M.DNA Marker DL2000;A.First round of Overlapping PCR;B.Second round of Overlapping PCR:lanes 3 and 4 are CRE1-tg1 and CRE1-tg2;C.Running gel after addition of the Bsa Ⅰ digestion site;D.Colony PCR.

Fig.6 Electrophoretic detection results of the connection between the target sgRNA expression box of CRE1 and pYLCRISPR/Cas9 vector M.DNA Marker DL2000;A.pYLCRISPR/Cas9 was digestion by BsaⅠ;B.Colony PCR.

Fig.7 Sequencing results of target sites on CRE1 double target knockout vector The target DNA sequence is labeled with shadow rectangles(the top is target site 1,bottom is target site 2).

Fig.8 Sequencing comparison results at target site 1 of CRE1 knockout Arabidopsis The red marker sequence part ATCCTCTCA CAACTCATTAC is the target site 1.

Fig.9 Comparison of seed germination potential of CRE1 gene mutants A.Germination potential of each CRE1 mutant;B.Seed germination phenotype of each CRE1 mutant.

References 22

[1]	李振华, 王建华. 种子活力与萌发的生理与分子机制研究进展[J]. 中国农业科学, 2015, 48(4):646-660.doi: 10.3864/j.issn.0578-1752.2015.04.03. doi: 10.3864/j.issn.0578-1752.2015.04.03
	Li Z H, Wang J H. Advances in research of physiological and molecular mechanism in seed vigor and germination[J]. Scientia Agricultura Sinica, 2015, 48(4):646-660.
[2]	Jameson P E, Song J C. Cytokinin:a key driver of seed yield[J]. Journal of Experimental Botany, 2016, 67(3):593-606.doi: 10.1093/jxb/erv461. doi: 10.1093/jxb/erv461 URL
[3]	Cortleven A, Leuendorf J E, Frank M, Pezzetta D, Bolt S, Schmülling T. Cytokinin action in response to abiotic and biotic stresses in plants[J]. Plant,Cell & Environment, 2019, 42(3):998-1018.doi: 10.1111/pce.13494. doi: 10.1111/pce.13494
[4]	Wang Y P, Shen W Z, Chan Z L, Wu Y. Endogenous cytokinin overproduction modulates ROS homeostasis and decreases salt stress resistance in Arabidopsis thaliana[J]. Frontiers in Plant Science, 2015, 6:1004.doi: 10.3389/fpls.2015.01004. doi: 10.3389/fpls.2015.01004
[5]	Wang Y P, Li L, Ye T T, Zhao S J, Liu Z, Feng Y Q, Wu Y. Cytokinin antagonizes ABA suppression to seed germination of Arabidopsis by downregulating ABI5 expression[J]. The Plant Journal, 2011, 68(2):249-261.doi: 10.1111/j.1365-313X.2011.04683.x. doi: 10.1111/j.1365-313X.2011.04683.x.
[6]	Huang X Z, Zhang X Y, Gong Z Z, Yang S H, Shi Y T. ABI4 represses the expression of type-A ARRs to inhibit seed germination in Arabidopsis[J]. The Plant Journal, 2017, 89(2):354-365.doi: 10.1111/tpj.13389. doi: 10.1111/tpj.13389 URL
[7]	Guan C M, Wang X C, Feng J, Hong S L, Liang Y, Ren B, Zuo J R. Cytokinin antagonizes abscisic acid-mediated inhibition of Cotyledon greening by promoting the degradation of ABSCISIC ACID INSENSITIVE5 protein in Arabidopsis[J]. Plant Physiology, 2014, 164(3):1515-1526.doi: 10.1104/pp.113.234740. doi: 10.1104/pp.113.234740 URL
[8]	Araújo S, Pagano A, Dondi D, Lazzaroni S, Pinela E, Macovei A, Balestrazzi A. Metabolic signatures of germination triggered by kinetin in Medicago truncatula[J]. Scientific Reports, 2019, 9(1):10466.doi: 10.1038/s41598-019-46866-6. doi: 10.1038/s41598-019-46866-6 URL
[9]	Inoue T, Higuchi M, Hashimoto Y, Seki M, Kobayashi M, Kato T, Tabata S, Shinozaki K, Kakimoto T. Identification of CRE1 as a cytokinin receptor from Arabidopsis[J]. Nature, 2001, 409(6823):1060-1063.doi: 10.1038/35059117. doi: 10.1038/35059117 URL
[10]	Vescovi M, Riefler M, Gessuti M, Novák O, Schmülling T, Lo Schiavo F. Programmed cell death induced by high levels of cytokinin in Arabidopsis cultured cells is mediated by the cytokinin receptor CRE1/AHK4[J]. Journal of Experimental Botany, 2012, 63(7):2825-2832.doi: 10.1093/jxb/ers008. doi: 10.1093/jxb/ers008 pmid: 22312114
[11]	Doroshenko A S, Danilova M N, Kudryakova N V, Soloviev A A, Kusnetsov V V. Cytokinin membrane receptors participate in regulation of plastid genome expression in the skotomorphogenesis[J]. Doklady Biochemistry and Biophysics, 2016, 469(1):294-297.doi: 10.1134/S1607672916040153. doi: 10.1134/S1607672916040153 pmid: 27599515
[12]	Franco-Zorrilla J M, Martin A C, Solano R, Rubio V, Leyva A, Paz-Ares J. Mutations at CRE1 impair cytokinin-induced repression of phosphate starvation responses in Arabidopsis[J]. The Plant Journal, 2002, 32(3):353-360.doi: 10.1046/j.1365-313x.2002.01431.x. doi: 10.1046/j.1365-313x.2002.01431.x.
[13]	Maruyama-Nakashita A, Nakamura Y, Yamaya T, Takahashi H. A novel regulatory pathway of sulfate uptake in Arabidopsis roots:Implication of CRE1/WOL/AHK4-mediated cytokinin-dependent regulation[J]. The Plant Journal, 2004, 38(5):779-789.doi: 10.1111/j.1365-313X.2004.02079.x. doi: 10.1111/j.1365-313X.2004.02079.x. URL
[14]	Tran L S P, Urao T, Qin F, Maruyama K, Kakimoto T, Shinozaki K, Yamaguchi-Shinozaki K. Functional analysis of AHK1/ATHK1 and cytokinin receptor histidine kinases in response to abscisic acid,drought,and salt stress in Arabidopsis[J]. PNAS, 2007, 104(51):20623-20628.doi: 10.1073/pnas.0706547105. doi: 10.1073/pnas.0706547105 URL
[15]	Jansen R, van Embden J D, Gaastra W, Schouls L M. Identification of genes that are associated with DNA repeats in prokaryotes[J]. Molecular Microbiology, 2002, 43(6):1565-1575.doi: 10.1046/j.1365-2958.2002.02839.x. doi: 10.1046/j.1365-2958.2002.02839.x. pmid: 11952905
[16]	Wang Y P, Wang J F, Guo S G, Tian S W, Zhang J, Ren Y, Li M Y, Gong G Y, Zhang H Y, Xu Y. CRISPR/Cas9-mediated mutagenesis of ClBG1 decreased seed size and promoted seed germination in watermelon[J]. Horticulture Research, 2021, 8(1):70.doi: 10.1038/s41438-021-00506-1. doi: 10.1038/s41438-021-00506-1 URL
[17]	Zhou J P, Yuan M Z, Zhao Y X, Quan Q, Yu D, Yang H, Tang X, Xin X H, Cai G Z, Qian Q, Qi Y P, Zhang Y. Efficient deletion of multiple circle RNA loci by CRISPR-Cas9 reveals Os06circ02797 as a putative sponge for OsMIR408 in rice[J]. Plant Biotechnology Journal, 2021, 19(6):1240-1252.doi: 10.1111/pbi.13544. doi: 10.1111/pbi.13544 URL
[18]	Shi S J, An L L, Mao J J, Aluko O O, Ullah Z, Xu F Z, Liu G S, Liu H B, Wang Q. The CBL-interacting protein kinase NtCIPK23 positively regulates seed germination and early seedling development in tobacco(Nicotiana tabacum L.)[J]. Plants(Basel), 2021, 10(2):323.doi: 10.3390/plants10020323. doi: 10.3390/plants10020323 URL
[19]	黎家, 李传友. 新中国成立70年来植物激素研究进展[J]. 中国科学(生命科学), 2019, 49(10):1227-1281.doi: 10.1360/SSV-2019-0197. doi: 10.1360/SSV-2019-0197
	Li J, Li C Y. Seventy-year major research progress in plant hormones by Chinese scholars[J]. Science in China, 2019, 49(10):1227-1281.
[20]	Han C, Yang P F. Studies on the molecular mechanisms of seed germination[J]. Proteomics, 2015, 15(10):1671-1679.doi: 10.1002/pmic.201400375. doi: 10.1002/pmic.201400375 URL
[21]	Dandekar T, Naseem M. Auxins and cytokinins in plant biology:methods and protocols[M]. New York: Springer New York, 2017.doi: 10.1007/978-1-4939-6831-2. doi: 10.1007/978-1-4939-6831-2
[22]	Nishiyama R, Watanabe Y, Fujita Y, Le D T, Kojima M, Werner T, Vankova R, Yamaguchi-Shinozaki K, Shinozaki K, Kakimoto T, Sakakibara H, Schmülling T, Tran L S P. Analysis of cytokinin mutants and regulation of cytokinin metabolic genes reveals important regulatory roles of cytokinins in drought,salt and abscisic acid responses,and abscisic acid biosynthesis[J]. The Plant Cell, 2011, 23(6):2169-2183.doi: 10.1105/tpc.111.087395. doi: 10.1105/tpc.111.087395 pmid: 21719693

Please choose a citation manager

Content to export