不同激素组合对番茄离体再生和相关基因表达的影响

doi:10.3969/j.issn.1004-1524.2022.09.08

浙江农业学报 ›› 2022, Vol. 34 ›› Issue (9): 1889-1900.DOI: 10.3969/j.issn.1004-1524.2022.09.08

不同激素组合对番茄离体再生和相关基因表达的影响

金宝霞(), 王伟杰, 朱晓林, 王贤, 魏小红()

甘肃农业大学生命科学技术学院,甘肃省作物遗传改良与种质创新重点实验室,甘肃省干旱生境作物学重点实验室,甘肃兰州, 730070

收稿日期:2022-01-07 出版日期:2022-09-25 发布日期:2022-09-30
通讯作者: 魏小红
作者简介:*魏小红,E-mail: weixh@gsau.edu.cn
金宝霞(1995—),女,甘肃金塔人,硕士研究生,研究方向为樱桃番茄抗黄化曲叶病毒病。E-mail: 1479342756@qq.com
基金资助:
国家自然科学基金(32060401);甘肃省优秀研究生“创新之星”(2021CXZX-404)

Effects of different hormone combinations on tomato in vitro regeneration and related gene expression

JIN Baoxia(), WANG Weijie, ZHU Xiaolin, WANG Xian, WEI Xiaohong()

Gansu Key Lab of Crop Genetic & Germplasm Enhancement, Gansu Provincial Key Lab of Aridland Crop Science, College of Life Science and Technology, Gansu Agricultural University, Lanzhou 730070, China

Received:2022-01-07 Online:2022-09-25 Published:2022-09-30
Contact: WEI Xiaohong

摘要/Abstract

摘要：

以樱桃番茄材料J6的子叶和下胚轴为试验对象,分别在激素组合A(2 mg·L^-1 6-BA+0/0.1/0.2/0.3 mg·L^-1 IAA,依次编号为A1、A2、A3、A4)、组合B(2 mg·L^-1 ZT+0/0.1/0.2/0.3 mg·L^-1 IAA,依次编号为B1、B2、B3、B4)的MS培养基上进行培养,研究不同激素组合对不同时期的番茄子叶和下胚轴脱分化、再分化情况,以及脱分化基因和再分化基因表达的影响。结果表明:外植体培养至第10天,子叶的脱分化率在所有处理下为100%,但脱分化基因的表达量相差较大;与B组合相比,A组合的下胚轴脱分化率更高,所有脱分化基因表达量也显著(P<0.05)上调。培养至第20 d,子叶愈伤组织在B组合下形态良好,脱分化基因Solyc01g091420.2.1、Solyc04g072900.1.1和Solyc04g054910.3.1表达量在B1和B2处理下显著上调;下胚轴愈伤形态在所有处理下疏松透亮,所有脱分化基因表达量只在B2处理下显著上调。培养至第60 d,子叶再分化率在B1和B2处理下最高,为67%和58%,再分化基因Solyc05g013540.1.1、Solyc02g022850.1.1和Solyc10g052510.2.1在B1和B2处理下高表达;下胚轴再分化率和基因表达量在所有处理下均较低。综上所述,番茄材料J6以子叶为外植体,在2 mg·L^-1 ZT+0/0.1 mg·L^-1 IAA的激素组合下离体再生效果最好,脱分化基因Solyc01g091420.2.1、Solyc04g072900.1.1、Solyc04g054910.3.1和再分化基因Solyc05g013540.1.1、Solyc02g022850.1.1、Solyc10g052510.2.1与子叶离体再生过程密切相关,从分子层面证实了不同激素组合处理对调控番茄不同类型外植体脱分化、再分化过程有重要影响。

关键词: 樱桃番茄, 脱分化, 再分化, 激素组合, 实时荧光定量PCR, 分子机制

Abstract:

Cotyledons and hypocotyls of cherry tomato material J6 were cultured on MS medium supplemented with hormone combinations A(2 mg·L^-1 6-BA+0/0.1/0.2/0.3 mg·L^-1 IAA, numbered A1, A2, A3, A4) and B (2 mg·L^-1 ZT+0/0.1/0.2/0.3 mg·L^-1 IAA, numbered B1, B2, B3, B4), respectively, so as to study the effects of different hormone combinations on the dedifferentiation and redifferentiation of cotyledons and hypocotyls at different stages, as well as the expression of dedifferentiation genes and redifferentiation genes. The results showed that the dedifferentiation rate of cotyledons was 100% in all treatments at the 10th day, but expression level of dedifferentiation genes differed greatly. Compared with combination B, the dedifferentiation rate of hypocotyls under combination A was higher and expression level of all dedifferentiation genes were also significantly (P<0.05) up-regulated. On the 20th day, callus morphology of cotyledon was better under combination B, and expression level of dedifferentiation genes Solyc01g091420.2.1, Solyc04g072900.1.1 and Solyc04g054910.3.1 were significantly up-regulated under treatment B1 and B2. The callus morphology of hypocotyl was loosed and bright under all treatments, and the expression level of all dedifferentiated genes were significantly up-regulated only under treatment B2.On the 60th day, the redifferentiation rate of cotyledons under treatment B1 and B2 reached 67% and 58%, and the redifferentiation genes Solyc05g013540.1.1, Solyc02g022850.1.1 and Solyc10g052510.2.1 were highly expressed. The redifferentiation rate and gene expression level in hypocotyls were lower in all treatments. In conclusion, cotyledons of J6 had the best regeneration effect in vitro under the treatment of 2 mg·L^-1 ZT+0/0.1 mg·L^-1 IAA. The dedifferentiation genes Solyc01g091420.2.1, Solyc04g072900.1.1 and Solyc04g054910.3.1, and the redifferentiation genes Solyc05g013540.1.1, Solyc02g022850.1.1 and Solyc10g052510.2.1 were closely related to the regeneration process. It was also confirmed that different hormone combinations had important effects on regulating the dedifferentiation and redifferentiation process of different types of tomato explants.

Key words: cherry tomato, dedifferentiation, redifferentiation, hormone combination, qRT-PCR, molecular mechanism

中图分类号:

S641.2

金宝霞, 王伟杰, 朱晓林, 王贤, 魏小红. 不同激素组合对番茄离体再生和相关基因表达的影响[J]. 浙江农业学报, 2022, 34(9): 1889-1900.

JIN Baoxia, WANG Weijie, ZHU Xiaolin, WANG Xian, WEI Xiaohong. Effects of different hormone combinations on tomato in vitro regeneration and related gene expression[J]. Acta Agriculturae Zhejiangensis, 2022, 34(9): 1889-1900.

图/表 11

表1 番茄脱分化与再分化基因引物信息

Table 1 Information of primers for dedifferentiated and redifferentiated genes of tomato

基因类型 Genetic types	基因注册号 Gene registration number	上游引物 Forward primer (5'→3')	下游引物 Reverse primer (5'→3')
脱分化基因	Solyc09g066260.3.1	GTCCTGCTAGATTTGCAGCCA	ACATCGATCGGGGACAGGAA
Dedifferentiated genes	Solyc06g083930.2.1	TCCTGTCTATGGTTGTGTTGCC	AAGCTTTGTGCTAACTGCCC
	Solyc01g091420.2.1	AGTGTGTTGCCGGTTGCATA	GGGTTGTTAGGTGGGCTTGT
	Solyc04g072900.1.1	CTAAGAACCGAACCCGCCTT	CCTTGTCGTAAGCCAGAGCA
	Solyc12g056980.1.1	CAATTGGGCTGAACCAAGCC	CGTTATGACGGAGATGGGGG
	Solyc04g054910.3.1	CCCAATGAAGCAAACGGGTG	AGCCAAGCTTTGGCAAATGG
再分化基因	Solyc05g013540.1.1	TGTACGAACAGCTGCCGAAT	TCATTTGCGGTAGCACCACT
Redifferentiated genes	Solyc03g082980.3.1	TCACAGACGTCCTGTTTCCG	GTACATTCACCCATGCCCCA
	Solyc12g042210.2.1	CTGCACGAGTATGGCTAGGG	TCCAACGACGTATTCGGTGA
	Solyc02g022850.1.1	GCAAGTCCAAGGGTGCAAAG	CGGTTCAACAACGCTTCTGG
	Solyc08g066840.3.1	TTGCTGTTGGAAGTAGCCGT	AGGTGCCCACCAGAAATACG
	Solyc10g052510.2.1	TCGAGCCTGCTAAAACAGCA	TTTCAATGGAACCGGGGCTT
内参基因Reference gene	UBI	TCGTAAGGAGTGCCCTAATGCTGAA	CAATCGCCTCCAGCCTTGTTGTA

图1 不同激素组合下子叶和下胚轴的脱分化情况 A,CK处理10 d的子叶;B,CK处理10 d的下胚轴;C,A2处理10 d子叶;D,B1处理10 d下胚轴;E,B1处理20 d子叶;F,B1处理20 d下胚轴。

Fig.1 Dedifferentiation of cotyledons and hypocotyls under different hormone combinations A, The cotyledons of 10 days under CK treatment; B, The hypocotyls of 10 days under CK treatment; C, The cotyledons of 10 days under A2 treatment; D, The hypocotyls of 10 days under B1 treatment; E, The cotyledons of 20 days under B1 treatment; F, The hypocotyls of 20 days under B1 treatment.

表2 不同激素组合对J6子叶、下胚轴脱分化率的影响

Table 2 Effects of different hormone combinations on dedifferentiation rate of cotyledon and hypocotyl of J6

处理 Treatments	外植体数 Number of explants	子叶脱分化率 Dedifferentiation rate of cotyledon/%		下胚轴脱分化率 Dedifferentiation rate of hypocotyl/%
处理 Treatments	外植体数 Number of explants	10 d	20 d	10 d	20 d
CK	60	0 b	0 b	0 f	0 e
A1	60	100 a	100 a	70.00±0.01 bc	71.67±2.89 b
A2	60	100 a	100 a	88.33±2.89 a	90.00±5.00 a
A3	60	100 a	100 a	78.33±7.64 ab	81.67±5.77 a
A4	60	100 a	100 a	81.67±5.77 ab	83.33±2.89 a
B1	60	100 a	100 a	35.00±5.00 e	36.67±2.89 d
B2	60	100 a	100 a	65.00±5.00 cd	66.67±2.89 bc
B3	60	100 a	100 a	56.67±2.89 d	58.33±5.77 c
B4	60	100 a	100 a	65.00±5.00 cd	66.67±2.89 bc

图2 不同激素处理下子叶和下胚轴的再分化情况 A,CK处理60 d的下胚轴;B,B1处理60 d的子叶;C,B4处理60 d的子叶;D,A3处理60 d的下胚轴。

Fig.2 Redifferentiation of cotyledons and hypocotyls treated with different hormone combinations A, The hypocotyls of 60 days under CK treatment; B, The cotyledons of 60 days under B1 treatment; C, The cotyledons of 60 days under B4 treatment; D, The hypocotyls of 60 days under A3 treatment.

表3 不同激素组合对J6子叶、下胚轴再分化率的影响

Table 3 Effects of different hormone combinations on the redifferentiation rate of cotyledon and hypocotyl of J6

处理 Treatments	外植体数量 Number of explants	子叶再分化率 Redifferentiation rate of cotyledon/%	下胚轴再分化率 Redifferentiation rate of hypocotyl/%
CK	60	0 d	0 d
A1	60	22.00±2.89 b	1.67±1.15 c
A2	60	13.00±2.89 bc	0 d
A3	60	0 d	15.00±0.01 a
A4	60	0 d	0 d
B1	60	67.00±2.89 a	1.67±0.58 c
B2	60	58.00±2.89 a	6.67±2.89 b
B3	60	15.00±0.58 bc	6.67±2.89 b
B4	60	3.00±0.58 c	1.67±0.01 c

图3 不同激素组合第10天子叶愈伤组织中脱分化基因的相对表达量编号1-6分别代表Solyc09g066260.3.1、Solyc06g083930.2.1、Solyc01g091420.2.1、Solyc04g072900.1.1、Solyc12g056980.1.1和Solyc04g054910.3.1。柱上无相同小写字母表示差异显著(P<0.05)。下同。

Fig.3 Relative expression level of dedifferentiated genes in callus of cotyledons treated with different hormone combinations at the 10th day 1-6 represented Solyc09g066260.3.1, Solyc06g083930.2.1, Solyc01g091420.2.1, Solyc04g072900.1.1, Solyc12g056980.1.1 and Solyc04g054910.3.1, respectively. Data marked without the same lowercase letter indicated significant differences at P<0.05. The same as below.

图4 不同激素处理第20天子叶愈伤组织中脱分化基因的相对表达量

Fig.4 Relative expression level of dedifferentiated genes in callus of cotyledons treated with different hormone combinations at the 20th day

图5 不同激素处理第10天下胚轴愈伤组织中脱分化基因的相对表达量

Fig.5 Relative expression level of dedifferentiated genes in callus of hypocotyls treated with different hormone combinations at the 10th day

图6 不同激素处理第20天下胚轴愈伤组织中脱分化基因的相对表达量

Fig.6 Relative expression level of dedifferentiated genes in callus of hypocotyls treated with different hormone combinations at the 20th day

图7 不同激素处理第60天子叶愈伤组织中再分化基因的相对表达量编号7-12分别代表Solyc05g013540.1.1、Solyc03g082980.3.1、Solyc12g042210.2.1、Solyc02g022850.1.1、Solyc08g066840.3.1和Solyc10g052510.2.1。下同。

Fig.7 Relative expression level of redifferentiation genes in callus of cotyledons treated with different hormone combinations at the 60th day 7-12 represented Solyc05g013540.1.1, Solyc03g082980.3.1, Solyc12g042210.2.1, Solyc02g022850.1.1, Solyc08g066840.3.1 and Solyc10g052510.2.1, respectively. The same as below.

图8 不同激素处理第60天下胚轴愈伤组织再分化基因的相对表达量

Fig.8 Relative expression level of redifferentiation genes in callus of hypocotyls treated with different hormone combinations at the 60th day

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不同激素组合对番茄离体再生和相关基因表达的影响

Effects of different hormone combinations on tomato in vitro regeneration and related gene expression

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