Page 149 - 《广西植物》2024年第5期
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5 期               刘芳等: Pre ̄miR172 及 miR172 调控油菜 AP2 基因表达的规律分析                               9 3 7

                 Abstract: The appropriate flowering time is of great significance for crop yield. In the cropping pattern of “rice ̄rice ̄oil”
                 in southern regionꎬ researching on the flowering period of Brassica napus can provide a theoretical basis for the breeding
                 of early maturing rape varieties. The AP2 family transcription factors in rape are widely involved in the growth and
                 development and play an important role during flower development. Howeverꎬ there are few studies exploring the
                 regulation of AP2 at the microRNA level. In order to investigate the regulatory functions of the miR172 precursor (pre ̄
                 miR72) and mature body (miR172) on AP2 gene in rapeꎬ the regulatory elements of miR172 and AP2 promoters were
                 predicted based on bioinformaticsꎬ then the evolutionary relationship of six rape AP2 genes and the targeting relationship
                 between miR172 and AP2 were analyzedꎬ and the expression patterns of AP2ꎬ miR172 and pre ̄miR172 in different
                 tissues of early and late maturing rape were detected by qRT ̄PCR. Finallyꎬ the correlation between miR172 abundance
                 and AP2 expression level was studiedꎬ as well as the correlation between pre ̄miR172 and miR172. The results were as
                 follows: (1) Both miR172 and AP2 promoter regions had cis ̄elements that regulated flower development. (2) The six
                 AP2 sequences holded the strong purification selectionꎬ and they were the target genes of miR172 based on their binding
                 sites for miR172. (3) miR172 family could promote the flowering of early maturing rape by increasing AP2 expression
                 levelsꎬ except for miR172d. In late maturing rapeꎬ miR172a and miR172c performed weakly in floweringꎬ while
                 miR172b and miR172d worked together to reduce the expression level of AP2 to inhibit flowering. (4) The pre ̄miR172
                 family had a promoting effect on the expression level of miR172 family in early maturing rapeꎻ in late maturing rapeꎬ
                 pre ̄miR172a and pre ̄miR172b exerted positive regulation on the formation of their mature bodiesꎬ while pre ̄miR172c
                 and pre ̄miR172d exerted the opposite effects. After overexpression of pre ̄miR172ꎬ the expression patterns of miR172
                 and AP2 remained consistent with the above resultsꎬ confirming the regulatory function of pre ̄miR172 on miR172 and
                 AP2. The results of this study enrich the functional regulation pathway of rape AP2 geneꎬ and provide new ideas for the
                 study of gene regulatory function.
                 Key words: rapeꎬ AP2ꎬ miR172ꎬ pre ̄miR172ꎬ expression pattern


                MicroRNA ( miRNA) 是一类内源性非编码小                   族中 OsIDS1 基因和 SNB 基因的表达ꎬ 诱导开花
            RNAꎬ一般长度为 18 ~ 36 个核苷酸ꎬ参与基因转录                      (Lee et al.ꎬ 2014)ꎮ Li 等(2019) 在大岩桐中发现
            水 平 的 调 控 ( 张 幸 媛 等ꎬ 2021)ꎮ 有 研 究 表 明ꎬ             miR172 过表达导致 SsAP2 ̄like 基因表达量下降ꎬ促
            miRNA 可以通过调控靶基因来调节植物生长发育                           使其提前开花ꎮ 此外ꎬmiR172 通过靶向 AP2 类基
            (Huijser & Schmidꎬ 2011)、开花时间(Spanudakis &         因可以调控花器官形态的建成ꎬ如藏红花(Tsaftaris
            Jacksonꎬ2014) 和逆境胁迫 ( Liu & Axtellꎬ 2015)ꎮ         et al.ꎬ 2012)、芥菜(Shivaraj & Singhꎬ 2016)、玉米
            miRNA 的形成先经过 RNA 聚合酶转录产生初级                         (Chuck et al.ꎬ 2007)、水稻(Hu et al.ꎬ 2009)、月季
            miRNA 前体(primary miRNA precusorꎬpri ̄miRNA)ꎬ        (Fran ois et al.ꎬ 2018)、苎麻(马鑫ꎬ2017) 等ꎬ可见
            再经 DCL1 切割后保留 70 ~ 500 个核苷酸序列形成                    miR172 ̄AP2 在花器官发育中发挥着重要作用(Ji et
            茎环二级结构ꎬ 即 miRNA 前体 ( miRNA precusorꎬ               al.ꎬ 2011)ꎮ
            pre ̄miRNA)ꎬ最后 pre ̄miRNA 通过相似剪切后形成                      AP2 亚家族是 AP2 / ERF 转录因子家族成员

            成 熟 miRNA ( Phillips et al.ꎬ 2007 )ꎮ 可 见 pre ̄      之一ꎬ包含 2 个重复的 AP2 保守结构域ꎬ主要参与
            miRNA 对于成熟体 miRNA 的形成具有重要意义ꎮ                       植物的生长发育过程( Trupiano et al.ꎬ 2013ꎻZhao
                 miR172 广泛存在于植物中ꎬ最早在拟南芥中被                      et al.ꎬ 2015ꎻ Neogy et al.ꎬ 2019)ꎮ 最早ꎬAP2 结构

            克隆出来ꎬ其对植物的生长发育起基础作用(Park et                        域在 拟 南 芥 中 发 现ꎬ 其 与 花 发 育 相 关 ( Jofuku et
            al.ꎬ 2002)ꎬ在植物的生殖转换、花发育、响应胁迫机                      al.ꎬ 1994 )ꎬ 此 后ꎬ 陆 续 在 水 稻 ( Nakano et al.ꎬ
            制等方面具有重要作用( 王幼宁等ꎬ2016ꎻ赵晓晖                          2006)、油菜( Ghorbani et al.ꎬ 2020)、苹果( Cheng
            等ꎬ2017)ꎮ 拟南芥中 miR172 过表达抑制 AP2 蛋白                  et al.ꎬ 2020)、葡萄(Licausi et al.ꎬ 2010)等多个物
            水平ꎬ同时抑制成花因子 TOE1 和 TOE2ꎬ导致拟南                       种中发现ꎬ其参与花发育过程ꎬ但功能不尽相同ꎮ
            芥提前开花(Aukerman & Sakaiꎬ 2003)ꎮ 在水稻中                AP2 的功能发挥会受到 miRNA 的调控ꎬ其调控开
            光敏色素可降低 miR172d 的表达ꎬ从而抑制 AP2 家                     花时间的机制往往有 miR172 的参与来实现ꎮ
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