龙眼体胚发生早期SAUR63/64基因克隆及表达分析
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国家自然科学基金(31672127);福建省自然科学基金(2020J01543);福建省高原学科建设经费(102/71201801101);福建农林大学创新基金(CXZX2019033S,CXZX2018078)


Cloning and Expression Analysis of SAUR63/64 during the Early Somatic Embryogenesis of Dimocarpus longan
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    摘要:

    SAUR(small auxin upregulated RNA)是一类生长素早期响应基因,与植物的生长发育密切相关。为揭示SAUR63和SAUR64在龙眼体胚发生早期的调控作用,该研究采用TA克隆龙眼SAUR63和SAUR64基因,对其进行亚细胞定位、qRTPCR表达分析和荧光原位杂交分析验证。结果显示:(1)龙眼SAUR63和SAUR64序列长度分别为386 bp和312 bp;系统发育树显示SAUR63、SAUR64与拟南芥亲缘关系较近;亚细胞定位结果显示SAUR63蛋白定位在细胞核与细胞外基质,SAUR64 蛋白定位在叶绿体。(2)预测发现SAUR63和SAUR64同时作为LTCONS_00038949、LTCONS_00038950及LTCONS_00038952(lnc38949、lnc38950及lnc38952)的靶基因。(3)qRTPCR检测发现SAUR63、SAUR64与3个lncRNAs均在不完全胚性紧实结构阶段高表达;SAUR63与3个lncRNAs在100和500 μmol·L-1赤霉素(GA3)处理下上调表达,且在500 μmol·L-1 GA3时表达量达到峰值; SAUR63、SAUR64、lnc38949和lnc38950均在35 ℃处理下被显著诱导表达,而lnc38950在35 ℃下受到抑制;此外,SAUR63、SAUR64和3个lncRNAs还响应SA、MeJA、盐胁迫和干旱胁迫的调控。(4)荧光原位杂交结果显示,SAUR63与lnc38949均没有定位在合子胚特定部位,且lnc38949表达量高于SAUR63。研究表明,SAUR63、SAUR64基因可能受到lncRNAs的调控,共同参与龙眼体胚发生早期的调控。

    Abstract:

    SAUR (small auxinup RNA) genes are a class of early response genes to growth hormone, which are closely related to plant growth and development. In order to reveal the regulatory roles of SAUR63 and SAUR64 during the early somatic embryogenesis of longan, we used TAcloning to obtain the coding sequences of SAUR63 and SAUR64, and then inserted into the vector for subcellular localization analysis, qRT PCR expression analysis and fluorescence in situ hybridization analysis. The results showed that: (1) the sequence length of SAUR63 and SAUR64 in longan were 386 and 312 bp, respectively. Phylogenetic tree analysis showed that SAUR63 and SAUR64 were highly homologous to Arabidopsis thaliana. Subcellular localization results showed that SAUR63 was localized in the nucleus and extracellular matrix, and SAUR64 was localized in the chloroplast. (2) SAUR63 and SAUR64 were both predicted to be as target genes of LTCONS_00038949, LTCONS_00038950 and LTCONS_00038952 (lnc38949, lnc38950 and lnc38952). (3) The results of qRT PCR showed that SAUR63, SAUR64 and three lncRNAs were highly expressed at the stage of incomplete embryonic compact structure. SAUR63 and three lncRNAs were upregulated under 100 and 500 μmol·L-1 gibberellin (GA3) treatment, and the expression level reached the peak under 500 μmol·L-1 GA3 treatment. SAUR63, SAUR64, lnc38949 and lnc38950 were significantly induced at 35 ℃, while lnc38950 was inhibited. In addition, SAUR63, SAUR64 and the three lncRNAs were also response to SA, MeJA, salt stress and drought stress. (4) Fluorescence in situ hybridization result showed that both SAUR63 and lnc38949 were not localized in the specific site of the zygotic embryo, and the expression of lnc38949 was higher than SAUR63. The results showed that SAUR63 and SAUR64 may be regulated by lncRNAs and participate in the regulation of longan early somatic embryogenesis.

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傅卓然,李卓蕴,陈 燕,等.龙眼体胚发生早期SAUR63/64基因克隆及表达分析[J].西北植物学报,2022,42(12):1991-2002

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  • 在线发布日期: 2023-01-15
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