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作物学报 ›› 2023, Vol. 49 ›› Issue (4): 988-995.doi: 10.3724/SP.J.1006.2023.24082

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

过表达LrAN2基因对马铃薯中花青素和糖苷生物碱含量的影响

李红艳1(), 李洁雅1, 李响1, 叶广继1,2,3,4,5,6, 周云1,2,3,4,5,6, 王舰1,2,3,4,5,6,*()   

  1. 1青海大学, 青海西宁 810016
    2青海省农林科学院, 青海西宁 810016
    3青海大学青藏高原生物技术教育部重点实验室, 青海西宁 810016
    4青海省马铃薯育种重点实验室, 青海西宁 810016
    5省部共建三江源生态与高原农牧业国家重点实验室, 青海西宁 810016
    6青藏高原种质资源研究与利用实验室, 青海西宁 810016
  • 收稿日期:2022-04-03 接受日期:2022-07-21 出版日期:2023-04-12 网络出版日期:2022-08-17
  • 通讯作者: *王舰, E-mail: jianwang2197@163.com
  • 作者简介:E-mail: 1286068155@qq.com
  • 基金资助:
    财政部和农业农村部国家现代农业产业技术体系建设专项(CARS-9);青海省科学自然基金项目-创新团队(2022-ZJ-902);青海省创新平台建设专项(2022-ZJ-Y01)

Effects of overexpression of LrAN2 gene on contents of anthocyanins and glycoalkaloids in potato

LI Hong-Yan1(), LI Jie-Ya1, LI Xiang1, YE Guang-Ji1,2,3,4,5,6, ZHOU Yun1,2,3,4,5,6, WANG Jian1,2,3,4,5,6,*()   

  1. 1Qinghai University, Xining 810016, Qinghai, China
    2Qinghai Academy of Agriculture and Forestry Sciences, Xining 810016, Qinghai, China
    3Qinghai University Key Laboratory of Qinghai-Tibet Plateau Biotechnology of the Ministry of Education, Xining 810016, Qinghai, China
    4Qinghai Provincial Key Laboratory of Potato Breeding, Xining 810016, Qinghai, China
    5Provincial and Ministry Co-construction of the State Key Laboratory of Sanjiangyuan Ecology and Plateau Agriculture and Animal Husbandry, Xining 810016, Qinghai, China
    6Laboratory for Research and Utilization of Qinghai Tibet Plateau Germplasm Resources, Xining 810016, Qinghai, China
  • Received:2022-04-03 Accepted:2022-07-21 Published:2023-04-12 Published online:2022-08-17
  • Contact: *E-mail: jianwang2197@163.com
  • Supported by:
    China Agriculture Research System of MOF and MARA(CARS-9);Qinghai Natural Science Foundation Program-Innovation Team(2022-ZJ-902);Qinghai Innovation Platform Construction Project(2022-ZJ-Y01)

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摘要：

目前对马铃薯(Solanum tuberosum L.)中MYB基因参与的花青素研究较为深入, 但对影响马铃薯品质和安全的糖苷生物碱(steroidal glycoalkaloids, SGAs)在不同组织中的变化规律及调控机制尚不完全清楚。LrAN2是黑果枸杞(Lycium ruthenicum)中的MYB基因, 与黑果枸杞果实中花青素的积累有关。本研究以野生型大西洋、2种转LrAN2大西洋株系(LrAN2oe#66和LrAN2oe#200)为试验材料, 检测不同组织中的花青素和SGAs含量, 并对SGAs生物合成相关基因的表达量分析。pH示差法检测花青素发现, 仅在转基因植株(LrAN2oe#200)叶片中检测到一定量的花青素(12 mg 100 g-1 FW)。高效液相色谱-串联质谱法检测SGAs发现, 3个材料不同组织SGAs含量变化为叶片>薯皮>薯肉。薯皮中的SGAs含量无显著性差异; 薯肉中LrAN2oe#66的SGAs含量较对照降低、LrAN2oe#200对照显著增加1.3倍, 但未超出安全标准(0.2 mg g-1 FW); 叶片中LrAN2oe#66的SGAs含量较对照增加1倍、LrAN2oe#200较对照显著增加3.8倍。qRT-PCR分析基因表达量发现, StHMG1和StSGT2基因受到LrAN2基因的调控在转基因植株中显著上调。本研究结果对马铃薯植株中花青素含量的积累具有指导意义, 为进一步解析马铃薯资源中花青素及SGAs的调控机制提供理论依据。

关键词: LrAN2基因, 马铃薯, 花青素, 糖苷生物碱

Abstract:

At present, the research on the anthocyanins involved in the MYB gene in potato (Solanum tuberosum L.) is relatively in-depth, but the change rule and regulatory mechanism of the glycoalkaloids (steroidal glycoalkaloids, SGAs) in different tissues, which affect the quality and safety of potato, are still unclear. LrAN2, the MYB gene in Lycium ruthenicum, is associated with the accumulation of anthocyanins in Lycium ruthenicum fruit. In this study, to detect the contents of anthocyanins and SGAs in different tissues, and to analyze the expression of genes related to SGA biosynthesis, wild-type Atlantic and two trans-LrAN2 Atlantic lines (LrAN2oe#66 and LrAN2oe#200) were used as the test materials. The detection of anthocyanins by pH differential method showed that only a certain number of anthocyanins (12 mg 100 g-1 FW) were detected in the leaves of transgenic plants (LrAN2oe#200). The contents of SGAs by HPLC-TMS in different tissues of the three materials were as follows: leaves>potato skin>potato flesh. There was no significant difference in the contents of SGAs in potato peels. Compared with the control, the SGAs content of LrAN2oe#66 in potato flesh was lower than the control, and the SGAs content of LrAN2oe#200 was significantly increased by 1.3 times, but did not exceed the safety standard (0.2 mg g-1 FW). The contents of SGAs in LrAN2oe#66 of leaves were increased by 1 times and LrAN2oe#200 was significantly increased by 3.8 times compared with the control. StHMG1 and StSGT2 genes were significantly up-regulated in transgenic plants under the regulation of LrAN2 gene by qRT-PCR. These results give a guiding significance for the accumulation of anthocyanins in potato plants and provide a theoretical basis for further analysis of the regulation mechanism of anthocyanins and SGAs in potato resources.

Key words: LrAN2 gene, potato, anthocyanin, glycoalkaloid

图1

马铃薯SGAs生物合成途径"

表1

试验所需引物序列"

引物名称
Primer name		 正向引物
Forward sequence (5'-3')		 反向引物
Reverse sequence (5'-3')
qStHMG1 CAGGTTCAAATGCAAGACTC GATAGATCTATTATATTTCAT
qStSQS1 ACTTGCAGAGACTCGGGAAC TCGGTTGCCAGAAAGTTGTG
qStCAS ACCATTACACTCTGCAGCAA GAACGATATTCTCCCAATGC
qStSSR2 AAGCGCCTTGAACAGAGGAA ACCCTTGACATTTGGCCCAT
qStGAME4 GGCTTGCATTTGAGGTGTTTA GAGCCTTGAGTCCCTTATGAT
qStGAME8a TGGGAGATATGACAATTC GCCATGCCAAAGTTATTACC
qStSGT1 GGAACAATCTCACTGCTC CACACACACACCAAGTTAC
qStSGT2 CAATCTTACCGCACTTATAG GTGTTTATTCCCAGCCCTAG
qStSMT1 GATATCTAGGGGACAGGT CAGGTAGCTTCTATTGCG
qStCYP51G AATGTGGGGTTGCTGTTAGT ATCAACCCACCAACAATAGG
Actin (internal reference gene) AGATGCTTACGCTGGATG GAATGC TTCCGGTGTGGTTGGATTCTGTTC

图2

部分过表达LrAN2基因马铃薯 M: DL 2000 marker; Plasmid: LrAN2质粒; WT: 野生型。"

图3

大西洋(Atlantic)和转基因马铃薯的表型特征(A)、大西洋和LrAN2oe#200的花青素含量(B) n.d.表示花青素含量为0 mg 100 g-1, 生物学重复n = 3。"

图4

Atlantic、LrAN2oe#66、LrAN2oe#200薯皮(A)、薯肉(B)和叶片(C)中的SGAs含量 不同小写字母表示不同品种间SGAs的差异显著性(P < 0.05)。"

图5

SGAs生物合成相关基因在Atlantic和LrAN2oe#200薯皮中的表达量变化 不同小写字母表示不同品种间基因表达量的差异显著性(P < 0.05)。"

图6

SGAs生物合成相关基因在Atlantic和LrAN2oe#200薯肉中的表达量变化 不同小写字母表示不同品种间基因表达量的差异显著性(P < 0.05)。"

图7

SGAs生物合成相关基因在Atlantic和LrAN2oe#200叶片中的表达量变化 不同小写字母表示不同品种间基因表达量的差异显著性(P < 0.05)。"

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