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Acta Agronomica Sinica ›› 2021, Vol. 47 ›› Issue (8): 1417-1426.doi: 10.3724/SP.J.1006.2021.01071

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS •     Next Articles

Cloning and expression analysis of lncRNA27195 and its target gene TaRTS in wheat (Triticum aestivum L.)

WANG Na1,2(), BAI Jian-Fang2, MA You-Zhi1, GUO Hao-Yu2, WANG Yong-Bo2, CHEN Zhao-Bo2, ZHAO Chang-Ping2,*(), ZHANG Ling-Ping2,*()   

  1. 1Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100087, China
    2Beijing Engineering and Technique Research Center for Hybrid Wheat, Beijing Academy of Agriculture and Forestry Sciences/Municipal Key Laboratory of the Molecular Genetics of Hybrid Wheat, Beijing 100097, China
  • Received:2020-09-02 Accepted:2021-01-13 Online:2021-08-12 Published:2021-02-22
  • Contact: ZHAO Chang-Ping,ZHANG Ling-Ping E-mail:wangna_00@sina.com;cp_zhao@vip.sohu.com;lpzhang8@126.com
  • Supported by:
    Natural Science Foundation of Beijing(6182014);Outstanding Scientist Cultivation Program of BAAFS(JKZX201907);Foundation for Youths of BAAFS(QNJJ201916);National Natural Science Foundation of China(31872881);Special Project of Science and Technology Innovation Ability Construction of BAAFS(KJCX20180403)

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Abstract:

Long non-coding RNA (lncRNA) is a non-coding RNA length over 200 bp, which is abundant in plants. It plays important roles in plant growth, development, and stress response by regulating gene expression or protein function. In the previous study, a fertility-related lncRNA named lncRNA27195 was screened and obtained by transcriptome sequencing from the anther of wheat Photoperiod-thermo Sensitive Genic Male Sterility (PTGMS) line BS366. To investigate the function of lncRNA27195 in wheat, the lncRNA27195 gene and its target gene TaRTS were cloned from BS366. Bioinformatics analysis were performed on TaRTS. The expressions of lncRNA27195 and TaRTS in different tissues and their expression correlation between them were analyzed by qRT-PCR. Meanwhile, the expression patterns of lncRNA27195 and TaRTS under different light and temperature treatments, and methyl jasmonate (MeJA) treatments were investigated. The results showed that the TaRTS gene with 315 bp length, encoded 104 amino acids. Additionally, RTS proteins were only found as anther-specific proteins in gramineae plants. Both lncRNA27195 and TaRTS with a significantly positive correlation were highly expressed in stamens, and revealed different expression patterns in different fertility environments. The results demonstrated that the expression of lncRNA27195 and TaRTS were also regulated by light and temperature. In addition, we found that the appropriate concentration of MeJA could promote the expression of lncRNA27195 and TaRTS while SA could inhibit the expression. The results indicated that under the induction of photoperiod, temperature, and plant hormones, IncRNA27195 positively regulated TaRTS gene expression, resulting in affecting pollen development and male fertility. This study contributed to the mechanism research and production application of PTGMS wheat.

Key words: lncRNA, RTS protein, anther, male sterility

Table 1

Primer information"

引物名称
Primer name		 引物序列
Primer sequence (5′-3′)		 产物大小
Product length (bp)
lncRNA27195-F TTTAATCTTCCGAGCTCACCCA 396
lncRNA27195-R TTTAAATGTTAAAACATGTTATATTTTTGA
TaRTS-F TTTAAATGGCACGCACCGGC 315
TaRTS-R TTTAATCAGAGCCCCTGGGTGGT
lncRNA27195-qRT-F GCATACGCACCGCATGTATAAT 109
lncRNA27195-qRT-R CTAGTCGCACAGGTCGACTGAA
TaRTS-qRT-F AGGTCGCATCGTGCCTCCT 114
TaRTS-qRT-R CCCCTGGGTGGTGCAGT
Actin1-F TTGGCATCTCTCAGCACCTTC 138
Actin1-R TCAGCCTTCGCAATCCACA

Fig. 1

Amplified fragment of lncRNA27195 and TaRTS (A) and sequence of lncRNA27195 (B), and TaRTS (C)"

Fig. 2

Comparison of the prokaryotic expression sequence of lncRNA27195 (A) and TaRTS (B)"

Fig. 3

The hydrophilicity (A), secondary structure (B), and tertiary structure (C) of TaRTS Blue line means alpha helix, green line means beta turn, red line means extended strand, and purple line means random coil."

Fig. 4

Subcellular localization of TaRTS in wheat protoplasts"

Fig. 5

Multiple alignments of protein sequences (A) and phylogenetic tree analysis (B) of TaRTS"

Table 2

The information of promoter elements"

上游启动子元件 Upstream promoter elements 功能 Function
CAAT-box, TATA-box 核心启动子元件 Core promoter element
ACE, G-box, GT1-motif, T-box, Sp1 光响应元件 Light responsiveness element
ABRE 脱落酸响应元件 Abscisic acid responsiveness element
CAT-box 分生组织表达相关元件 Related to meristem expression element
CCAAT-box MYBHv1结合位点 MYBHv1 binding site
CGTCA-motif, TGACG-motif 茉莉酸甲酯响应元件 MeJA-responsiveness responsiveness element
LTR 低温响应元件 Low-temperature responsiveness element
MSA-like 细胞周期调控元件 Cell cycle regulation element

Fig. 6

Relative expression levels of lnc27195 (A) and TaRTS (B) in different tissues"

Fig. 7

Relative expression levels of TaRTS (A), lncRNA27195 (B), and the correlation analysis (C) of TaRTS and lncRNA27 195 * means significantly different at P<0.05, and ** means highly significant difference at P<0.01."

Fig. 8

Relative expression levels of TaRTS and lncRNA27195 in different environments T1: long-light environment with high temperature (20℃, 14 h L/10 h D); T2: short-light environment with high temperature (20℃, 12 h L/12 h D); T3: long-light environment with low temperature (12℃, 14 h L/10 h D); T4: short-light environment with low temperature (12℃, 12 h L/12 h D). * means significantly different at P < 0.05."

Fig. 9

Relative expression levels of TaRTS and lncRNA27195 under MeJA and SA treatments"

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