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Acta Agronomica Sinica ›› 2021, Vol. 47 ›› Issue (6): 1054-1069.doi: 10.3724/SP.J.1006.2021.04078

• SPECIAL SECTION: GENOMICS AND GENETIC IMPROVEMENT IN MAIN BAST FIBER CROPS • Previous Articles     Next Articles

Genome-wide identification of GRAS transcription factor and expression analysis in response to cadmium stresses in hemp (Cannabis sativa L.)

YIN Ming(), YANG Da-Wei, TANG Hui-Juan, PAN Gen, LI De-Fang, ZHAO Li-Ning, HUANG Si-Qi*()   

  1. Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences/Key Laboratory of Biological and Processing for Bast Fiber Crops, Ministry of Agriculture and Rural Affairs, Changsha 410205, Hunan, China
  • Received:2020-03-27 Accepted:2020-11-13 Online:2021-06-12 Published:2020-12-28
  • Contact: HUANG Si-Qi E-mail:1586887698@qq.com;huangsiqi@caas.cn
  • Supported by:
    The China Agriculture Research System(CARS-16-E02)

Abstract:

GRAS transcription factors play important roles in plant growth, development and stress responses. In order to systematically analyze the GRAS transcription factor family in hemp, we performed a genome-wide identification of hemp GRAS genes, analyzed their physical and chemical properties, phylogenetic development, gene structure, and GRAS gene expression under cadmium stress in two hemp varieties (Yunma 1 and Inner Mongolia Xiaolidama). The results showed that there were 54 GRAS transcription factors in hemp genome, with the protein length from 415 to 757, encoding 96.30% of acidic proteins, the molecular weight from 46,405.05 to 85,748.52 kD and the isoelectric point from 4.77 to 8.54. These transcription factors were divided into nine subfamilies, among which, PAT1, LS, SHR, and HAM were more conserved, PAT1, LISCL, and CsGRASA had a large number of tandem repeats of genes, and CSGRAS12 genes were all present in collinearity analysis of five plants. Yunma 1 and Inner Mongolia Xiaolidama were treated with cadmium stress. Plant height and the fresh weight decreased in Yunma 1 and Inner Mongolia Xiaolidama, by 10.48%, 6.33% and 66.07%, 42.67%, respectively, indicating that Yunma 1 was more tolerant than Inner Mongolia Xiaolidama under cadmium stress. Among 54 GRAS genes in Yunma 1, 42 genes (77.78%) were up-regulated by 1.05-18.10, and 11 genes (20.37%) were down-regulated by 0.13-0.91. Among 54 GRAS genes in small-grain cannabis in Inner Mongolia, 27 genes (50.00%) were up-regulated by 1.01-6.46, and 27 genes (50.00%) were down-regulated by 0.30-0.96. This study showed that the 40 homologous GRAS gene in Yunma 1 were significanlty up- or down-regulated than those in Inner Mongolia Xiaolidama under cadmium stress, indicating that these GRAS genes were significantly related to cadmium stress. This study can provide a reference for subsequent mining and verification of GRAS genes in hemp.

Key words: hemp, GRAS transcription factors, phylogenetic analysis, gene structure analysis, expression analysis

Table 1

GRAS genes and their related information"

基因名称
Gene name
NCBI基因登录号
NCBI gene accession
蛋白长度
Length (aa)
等电点
pI
分子量
Molecular weight (kD)
CsGRAS1 LOC115696991 458 4.77 52,006.96
CsGRAS2 LOC115698980 494 5.15 54,901.55
CsGRAS3 LOC115698988 724 5.99 81,047.24
CsGRAS4 LOC115700662 455 5.66 49,596.06
CsGRAS5 LOC115701004 454 6.68 50,178.21
CsGRAS6 LOC115702418 528 5.24 57,541.56
CsGRAS7 LOC115703632 559 6.21 62,215.78
CsGRAS8 LOC115705736 722 5.77 78,760.91
CsGRAS9 LOC115705815 722 5.72 78,817.91
CsGRAS10 LOC115705915 721 5.74 78,730.89
CsGRAS11 LOC115706185 539 5.51 59,352.41
CsGRAS12 LOC115707313 544 6.33 61,258.62
CsGRAS13 LOC115710202 582 4.92 64,716.73
CsGRAS14 LOC115710247 582 4.92 64,716.73
CsGRAS15 LOC115710247 582 4.92 64,716.73
CsGRAS16 LOC115710247 582 4.92 64,716.73
CsGRAS17 LOC115710247 582 4.92 64,716.73
CsGRAS18 LOC115710247 582 4.92 64,716.73
CsGRAS19 LOC115710247 582 4.92 64,716.73
CsGRAS20 LOC115710262 514 4.93 57,833.13
CsGRAS21 LOC115711337 467 5.82 52,554.66
CsGRAS22 LOC115711473 549 6.13 61,322.10
CsGRAS23 LOC115712444 497 5.28 56,735.16
CsGRAS24 LOC115712525 504 5.68 56,355.13
CsGRAS25 LOC115712712 613 5.08 67,527.83
CsGRAS26 LOC115712875 508 6.30 56,609.53
CsGRAS27 LOC115714404 622 5.33 68,325.01
CsGRAS28 LOC115714408 600 5.95 66,439.36
CsGRAS29 LOC115715793 628 5.08 71,316.90
CsGRAS30 LOC115716239 617 6.03 68,009.17
CsGRAS31 LOC115716331 700 5.99 76,887.41
CsGRAS32 LOC115716409 616 6.19 67,882.02
CsGRAS33 LOC115716431 752 6.29 82,128.85
CsGRAS34 LOC115717012 697 5.36 77,185.61
CsGRAS35 LOC115718811 645 6.73 73,634.61
CsGRAS36 LOC115718865 746 5.80 85,106.83
CsGRAS37 LOC115718865 709 5.66 80,996.82
CsGRAS38 LOC115718866 630 7.24 72,454.10
CsGRAS39 LOC115718867 621 5.69 71,491.35
CsGRAS40 LOC115718867 551 6.37 63,913.20
CsGRAS41 LOC115718867 506 8.54 58,824.80
CsGRAS42 LOC115719316 749 5.03 84,965.93
CsGRAS43 LOC115719656 757 5.10 85,748.52
CsGRAS44 LOC115719656 636 5.20 73,211.22
基因名称
Gene name
NCBI基因登录号
NCBI gene accession
蛋白长度
Length (aa)
等电点
pI
分子量
Molecular weight (kD)
CsGRAS45 LOC115719730 698 5.68 78,688.43
CsGRAS46 LOC115722004 577 4.94 66,178.24
CsGRAS47 LOC115723037 575 5.14 64,688.92
CsGRAS48 LOC115723133 436 5.58 49,164.54
CsGRAS49 LOC115723500 454 6.00 51,420.59
CsGRAS50 LOC115724710 577 6.05 64,503.06
CsGRAS51 LOC115724919 548 5.14 61,008.73
CsGRAS52 LOC115724919 415 5.20 46,405.05
CsGRAS53 LOC115725148 463 5.64 52,596.41
CsGRAS54 LOC115725491 756 6.16 85,229.91

Fig. 1

Phylogenetic tree of GRAS family in hemp and Arabidopsis Different colors indicate different subfamily, and different genes under the same color belong to the same subfamily."

Fig. 2

Gene structure analysis of GRAS genes in hemp"

Fig. 3

Gene motifs of GRAS in hemp"

Fig. 4

Chromosomal locations of GRAS genes in hemp"

Fig. 5

GRAS synteny analysis among chromosomes in hemp"

Fig. 6

Synteny analysis of GRAS genes between Gossypium raimondii, Cannabis sativa L., and Oryza sativa L."

Fig. 7

Synteny analysis of GRAS genes between Glycine max, Cannabis sativa L., and Zea mays L."

Table 2

Relative expression of GRAS genes in Yunma 1"

基因名称
Gene name
云麻1号对照组
Yunma 1
control check
云麻镉胁迫组
Yunma 1
cadmium stress
变化值
Fold change
基因名称
Gene name
云麻对照组
Yunma 1
control check
云麻镉胁迫组
Yunma 1
cadmium stress
变化值
Fold change
CsGRAS24 2.44 0.33 0.13* CsGRAS28 2.31 4.07 1.76*
CsGRAS46 4.02 0.58 0.14* CsGRAS13 4.74 9.76 2.06*
CsGRAS25 42.77 20.24 0.47* CsGRAS32 13.12 27.08 2.06*
CsGRAS33 6.56 3.12 0.48* CsGRAS52 8.33 18.67 2.24*
CsGRAS2 1.62 0.78 0.48 CsGRAS1 0.21 0.46 2.24
CsGRAS11 30.07 14.91 0.50* CsGRAS29 0.21 0.46 2.24
CsGRAS22 1.63 0.91 0.56 CsGRAS12 19.52 44.15 2.26*
CsGRAS54 6.20 4.59 0.74 CsGRAS30 7.67 18.41 2.40*
CsGRAS6 0.32 0.28 0.88 CsGRAS5 0.37 0.97 2.65
CsGRAS42 9.74 8.87 0.91 CsGRAS7 1.00 2.68 2.69*
CsGRAS48 0.25 0.26 1.05 CsGRAS38 2.22 6.10 2.75*
CsGRAS9 6.16 6.88 1.12 CsGRAS43 0.74 2.10 2.85*
CsGRAS4 8.31 9.43 1.14 CsGRAS47 0.04 0.11 2.91
CsGRAS21 2.66 3.16 1.19 CsGRAS40 0.31 0.99 3.19
CsGRAS20 0.76 0.93 1.21 CsGRAS17 3.35 11.74 3.51*
CsGRAS10 7.49 9.54 1.27 CsGRAS37 12.42 61.52 4.95*
CsGRAS36 8.79 11.82 1.34 CsGRAS41 0.21 1.19 5.59*
CsGRAS31 14.46 19.51 1.35 CsGRAS26 0.04 0.21 5.64
CsGRAS23 0.20 0.27 1.37 CsGRAS50 8.33 57.94 6.96*
基因名称
Gene name
云麻1号对照组
Yunma 1
control check
云麻镉胁迫组
Yunma 1
cadmium stress
变化值
Fold change
基因名称
Gene name
云麻对照组
Yunma 1
control check
云麻镉胁迫组
Yunma 1
cadmium stress
变化值
Fold change
CsGRAS15 3.04 4.43 1.46 CsGRAS3 0.01 0.14 10.50*
CsGRAS49 0.97 1.44 1.49 CsGRAS39 0.03 0.60 18.10*
CsGRAS8 8.98 13.70 1.53* CsGRAS18 0.00 0.30 0.30*
CsGRAS27 18.49 28.66 1.55* CsGRAS19 0.00 0.17 0.17*
CsGRAS44 4.33 6.75 1.56 CsGRAS34 0.00 0.03 0.03
CsGRAS16 0.32 0.51 1.59 CsGRAS35 0.00 0.32 0.32*
CsGRAS14 0.76 1.24 1.62* CsGRAS45 0.00 0.00 0.00
CsGRAS51 6.82 11.11 1.63* CsGRAS53 0.27 0.00 -0.27*

Table 3

Relative expression of GRAS genes in Inner Mongolia Xiaolidama"

基因名称
Gene name
内蒙大麻对照组
Inner Mongolia Xiaolidama
control check
内蒙大麻镉胁迫组
Inner Mongolia Xiaolidama
cadmium stress
变化值
Fold change
基因名称
Gene name
内蒙大麻对照组
Inner Mongolia Xiaolidama
control check
内蒙大麻镉胁迫组
Inner Mongolia Xiaolidama
cadmium stress
变化值
Fold change
CsGRAS24 5.40 1.61 0.30* CsGRAS31 20.57 20.70 1.01
CsGRAS53 0.84 0.37 0.44* CsGRAS40 0.60 0.62 1.02
CsGRAS22 2.70 1.35 0.50* CsGRAS39 0.31 0.33 1.05
CsGRAS48 0.34 0.17 0.50 CsGRAS42 9.72 10.66 1.10
CsGRAS25 49.36 28.23 0.57* CsGRAS4 8.38 9.73 1.16
CsGRAS21 2.54 1.61 0.63 CsGRAS44 4.87 5.91 1.21
CsGRAS33 12.12 7.76 0.64* CsGRAS52 12.38 15.14 1.22
CsGRAS36 10.98 7.07 0.64 CsGRAS13 6.41 8.78 1.37
CsGRAS41 1.27 0.97 0.76 CsGRAS47 0.08 0.11 1.38
CsGRAS17 8.55 6.64 0.78 CsGRAS28 1.85 2.58 1.39
CsGRAS11 37.71 29.54 0.78 CsGRAS30 12.06 17.68 1.47*
CsGRAS18 0.22 0.18 0.78* CsGRAS9 6.75 9.90 1.47*
CsGRAS20 0.28 0.23 0.79 CsGRAS51 14.04 21.05 1.50*
CsGRAS7 1.70 1.39 0.82 CsGRAS38 2.68 4.34 1.62
CsGRAS6 0.28 0.23 0.82 CsGRAS12 17.93 31.35 1.75*
CsGRAS1 0.48 0.40 0.83 CsGRAS15 1.82 3.65 2.00
CsGRAS29 0.48 0.40 0.83 CsGRAS45 0.04 0.10 2.73
CsGRAS14 1.08 0.94 0.87 CsGRAS16 0.10 0.30 3.00
CsGRAS27 20.63 18.12 0.88 CsGRAS50 13.12 41.91 3.19*
CsGRAS23 0.29 0.26 0.89 CsGRAS3 0.01 0.05 3.38
CsGRAS49 1.10 0.99 0.89 CsGRAS5 0.31 1.20 3.87*
CsGRAS32 13.96 12.66 0.91 CsGRAS37 8.00 34.34 4.29*
CsGRAS8 14.56 13.35 0.92 CsGRAS43 2.10 9.65 4.59*
CsGRAS10 4.19 3.87 0.92 CsGRAS26 0.08 0.50 6.46*
CsGRAS54 7.36 6.96 0.94 CsGRAS19 0.00 0.17 0.17*
CsGRAS46 1.17 1.12 0.95 CsGRAS34 0.00 0.23 0.23*
CsGRAS2 2.82 2.71 0.96 CsGRAS35 0.00 0.89 0.89*

Fig. 8

Growth status of two hemp varieties under normal and cadmium stress environment"

Fig. 9

Relative expression of GRAS homologous genes"

Fig. 10

qPCR expression of 12 genes *, **, *** mean significant differences at the 0.05, 0.01, and 0.001 probability levels, respectively."

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