Crops ›› 2021, Vol. 37 ›› Issue (1): 16-25.doi: 10.16035/j.issn.1001-7283.2021.01.003

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Cloning, Molecular Characteristics Analysis and Genetic Transformation of Arabidopsis thaliana of ZmGS5 Gene in Maize

Liu Xiaoli1,2(), Han Litao1, Wei Nan1, Shen Fei1, Cai Yilin2()   

  1. 1Department of Food Engineering, Henan Vocational College of Agriculture, Zhengzhou 451450, Henan, China
    2Maize Research Institute, Southwest University, Chongqing 400715, China
  • Received:2020-07-30 Revised:2021-01-12 Online:2021-02-15 Published:2021-02-23
  • Contact: Cai Yilin E-mail:314496088@qq.com;caiyilin1789@163.com

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Abstract:

High yield is an important goal of the maize breeding, grain size is a major determinative factor of high yield. In this study, based on the method of homology cloning, the OsGS5 sequence was used as a template to obtain the ZmGS5 gene. The sequencing result revealed that the full length cDNA of ZmGS5 was 1695bp. The Open Reading Frame (ORF) encoding 496 amino acid was 1491bp. The ZmGS5 protein contained conservative structure domain Peptidase-S10, a signal peptide, an active site of the serine carboxypeptidases, which were all conformed to the structure of serine carboxypeptidase family and also conformed to the OsGS5 related research results. Phosphorylation locus analysis indicated that the protein contained recognition loci of Ser, Thr, Tyr kinases. The result of real-time quantitative PCR showed that ZmGS5 had a much higher expression level in tassel and leaves, and the lower expression level in embryo and endosperm. According to agrobacterium-mediated method, we established genetic transformation system in Arabidopsis and obtained homozygous transgenic lines. The 1000-seed weight of T3 Arabidopsis (0.0169g) was higher than that of wild type (0.0139g).

Key words: ZmGS5, Gene cloning, Serine carboxypeptidases, Molecular characteristics analysis, Genetic transformation

Table 1

Sequences of all PCR primers used"

引物Primer 引物序列Primer sequence 引物Primer 引物序列Primer sequence
ZmGS5-F CATATGGCGACAATGGCAGGG ZmGS5-R TTGTCATCTGTGTGTGGGAAGC
ZmGS5-3′-GSP1 TGCTGTTGACAAGGCAGTCGTAGCCAA ZmGS5-3′-GSP2 CGTAGCCAACAGACAGGAACATTTCAG
ZmGS5-5′-GSP1 GATGATGTGTTGGTGTAGGAGAAGCCA ZmGS5-5′-GSP2 ATTGAGCCAGAGTAGGAGAGGCTTGTG
UPM CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT NUP CTAATACGACTCACTATAGGGC
ZmGS5-qF CACAAGCCTCTCCTACTCTGGCTCAAT ZmGS5-qR TTCGTGGCTCCTATACTGCGGAAACCT

Fig.1

Rapid amplification of 3′ and 5′ ends of ZmGS5 1: 3′-RACE, 2: 5′-RACE, M: marker"

Fig.2

Amino acid composition of ZmGS5"

Fig.3

ProtScale analysis of ZmGS5"

Fig.4

Secondary structure of different plants homologous proteins of ZmGS5"

Fig.5

Spatial structure of ZmGS5 protein"

Table 2

Confidently predicted signal peptides, repeats, and domains of OsGS5 and ZmGS5"

蛋白
Protein		 名称
Name		 起点
Starting point		 终点
End point		 E值
E-value
OsGS5 信号肽 1 30 N/A
简单重复组件 2 14 N/A
简单重复组件 17 28 N/A
Peptidase-S10结构域 48 475 1.1e-136
ZmGS5 信号肽 1 37 N/A
简单重复组件 13 25 N/A
Peptidase-S10结构域 64 489 8.2e-141
简单重复组件 313 330 N/A

Fig.6

Prediction of signal peptides of ZmGS5"

Table 3

Comparison of ZmGS5 and OsGS5 in phosphorylation sites"

名称
Name		 位点
Site		 氨基酸识别位点
Amino acid recognition site		 值
Value		 名称
Name		 位点
Site		 氨基酸识别位点
Amino acid recognition site		 值
Value		 预测
Forecast
ZmGS5 73 SPPVSQFAG 0.939 OsGS5 57 SPAVSQFAG 0.622 丝氨酸Senine
99 EAQTSPAHK 0.654 83 EAQASPAPE 0.844
163 GVGFSYTNT 0.780 149 GVGFSYTNT 0.502
205 EFYISGESY 0.524 191 EFYISGESY 0.610
296 SAVFSQYQE 0.662 283 NIIFSQYNQ 0.602
344 IRMFSGYDP 0.997 328 IKMFSGYDP 0.995
ZmGS5 167 SYTNTSSDL 0.748 OsGS5 153 SYTNTSSDL 0.748 苏氨酸Threonine
463 MTMVTIRGA 0.815 449 MTMVTVRGA 0.769
ZmGS5 184 AEDAYSFLV 0.902 OsGS5 170 AEDAYSFLV 0.902 酪氨酸Tyrosine
203 SHEFYISGE 0.821 189 DNEFYISGE 0.925
209 SGESYAGHY 0.936 195 SGESYAGHY 0.936
222 AELVYDRNK 0.865 208 ADLVYERNK 0.842
248 LTDDYYDSK 0.906 235 LTDDYYDSK 0.906
249 TDDYYDSKG 0.980 236 TDDYYDSKG 0.980
269 SDEVYERIK 0.986 256 SDQVYERIK 0.952
350 YDPCYSSNA 0.970 334 YDPCYSSYA 0.917
357 NAEKYFNDA 0.948 341 YAEDYFNKH 0.955

Fig.7

The homology analysis of ZmGS5 and GS5 in other plants"

Table 4

The change of allelic variation in cDNA sequences of ZmGS5 and amino acid sequence of ZmGS5"

多态性位点
Polymorphic locus		 43、44、45 68 132 151 241 421 673 690 757 808 879 1231 1304 1357 1394 1420 1456 1457
cDNA突变位点
cDNA mutation		 插入或删除
Insert or delete		 T→C C→T C→T C→T G→A C→T C→T A→C C→T G→T G→C C→T C→A A→G T→C C→T A→G
B73 T C C C G C C A T G G C C A T C A
Mo17 T C C C G C C A T T G C C A T C A
178 T C C C G C C A T G G C C A T C A
郑58 T C C C G T C C C G C T A G C T G
昌7-2 GTC C C T T A T C C C G C T A G C T G
黄C GTC C C T T A T T C C G G C C A T C A
I15016 GTC C T T T A T T C C G G C C A T C A
I15018 T C C C G C C A T G G C C A T C A
I15020 T C C C G T C C C G C T A G C T G
I15030 GTC C C T T A T T C C G G C C A T C A
I15031 GTC C C T T A T T C C G G C C A T C A
氨基酸突变位点
Amino acid mutation		 插入或删除
Insert or delete		 F→L A→V T→I S→I I→V T→A
B73 F A T S I T
Mo17 F A T I I T
178 F A T S I T
郑58 F A T S V A
昌7-2 S L A T S V A
黄C S L A I S I T
I15016 S L V I S I T
I15018 F A T S I T
I15020 F A T S V A
I15030 S L A I S I T
I15031 S L A I S I T

Fig.8

Alignment of the amino acid sequence"

Fig.9

Neighbor-joining tree of SCPL family proteins"

Fig.10

The qRT-PCR analysis of ZmGS5 gene expression in different tissues of maize"

Fig.11

Kernel phenotypes of eleven inbred lines"

Table 5

Phenotypic analysis of eleven inbred lines kernels"

指标Index B73 Mo17 178 郑58 Zheng 58 昌7-2 Chang 7-2 黄C Huang C I15016 I15018 I15020 I15030 I15031
百粒重100-grain weight (g) 19.14 28.28 18.99 24.56 23.78 24.77 18.04 19.05 18.52 24.98 24.54
百粒体积100-grain volume (mL) 18.50 24.67 17.33 23.83 21.00 22.50 17.05 18.30 17.93 22.53 23.40
十粒长10-grain length (cm) 9.90 9.92 7.65 9.86 9.55 9.98 7.69 7.85 7.64 10.25 10.30
十粒宽10-grain width (cm) 7.03 8.51 7.61 7.85 7.38 7.71 7.45 7.49 7.57 7.50 7.58
十粒厚10-grain thickness (cm) 5.07 6.01 5.96 5.81 5.94 5.63 5.66 5.89 5.92 5.32 5.28

Fig.12

The detection of ZmGS5 in the leaves of T0 generation"

Fig.13

Screening of the T1 and T2 generations of transgenic Arabidopsis"

Table 6

The 1000-seed weight of the wild-type plants and T3 plants of Arabidopsis"

编号
No.		 千粒重
1000-seed
weight (g)		 编号
No.		 千粒重
1000-seed
weight (g)		 编号
No.		 千粒重
1000-seed
weight (g)
WT-1 0.0139 WT-2 0.0132 WT-3 0.0140
WT-5 0.0139 WT-6 0.0143
T3-7-1 0.0172 T3-3-1 0.0189 T3-1-10 0.0175
T3-1-11 0.0181 T3-7-9 0.0170 T3-14-10 0.0184
T3-1-6 0.0187 T3-10-13 0.0167 T3-7-7 0.0150
T3-13-11 0.0151 T3-1-9 0.0192 T3-2-16 0.0147
T3-8-5 0.0176 T3-7-11 0.0152 T3-18-3 0.0150
T3-2-3 0.0164 T3-20-8 0.0182 T3-14-5 0.0192
T3-10-7 0.0168 T3-5-9 0.0159 T3-2-17 0.0182
T3-19-1 0.0156 T3-1-4 0.0168 T3-20-6 0.0151
T3-14-1 0.0181 T3-13-14 0.0177 T3-3-2 0.0157
T3-2-9 0.0168 T3-3-4 0.0187 T3-6-3 0.0162
T3-19-2 0.0155 T3-4-6 0.0183 T3-8-10 0.0150
T3-19-5 0.0180 T3-14-9 0.0153 T3-10-13 0.0170
T3-13-8 0.0183
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