Over-expression of JcSEUSS1 from Jatropha curcas Induces the Accumulation of Anthocyanin in Leaves

Wang Jingxian ^{1, 2}　Ming Xin ^{1, 2}　Tao Yanbin ²　Tang Mingyong ^{2 *}　Xu Zengfu ^2*

1 School of Life Sciences, University of Science and Technology of China, Hefei, 230027; 2 Key Laboratory of Tropical Plant Resources and Sustainable Use, Innovative Academy of Seed Design, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Menglun, 666303

* Co-corresponding authors, tangmingyong@xtbg.ac.cn; zfxu@xtbg.ac.cn

Abstract　Anthocyanins are a class of important secondary metabolites in plants and play important roles in photoprotection and antioxidation. In this study, we cloned the JcSEUSS1 gene from Jatropha curcas. Sequence alignment indicates JcSEUSS1 contains a highly conserved LIM domain, and the sequence of JcSEUSS1 is similar to other SEUSS proteins. The expression pattern of JcSEUSS1 in different organs of Jatropha plants was analyzed by real-time fluorescence quantitative PCR, and it was found that the gene was expressed in all tissues. Furthermore, JcSEUSS1 over-expression significantly promoted the accumulation of anthocyanin in leaves of Jatropha. The expression of chalketone synthase (JcCHS) and chalketone isomerase (JcCHI), which are involved in anthocyanin biosynthesis, were significantly up-regulated in JcSEUSS1-transgenic lines. Our results indicate that JcSEUSS1 may positively regulate the anthocyanin synthesis in leaves of Jatropha.

Keywords　Jatropha curcas; JcSEUSS1; Over-expression; Anthocyanin

花青素(Anthocyanins)广泛存在于植物的各个组织部位，是一种水溶性的黄酮类色素(Cuyckens and Claeys, 2004)；有利于植物抗氧化和吸引昆虫授粉，还可用于制成抗癌性药物(Holton and Cornish 1995; Wu et al., 2004; Li et al., 2007)。目前，花青素合成代谢途径在拟南芥和玉米等模式植物中研究较为清楚。其代谢途径大致分为三个阶段：第一阶段是花青素合成前体苯丙氨酸在苯丙氨酸裂解酶(phenylalanine ammonia, PAL)、肉桂酸羟化酶(cinnamate-4-hydroxylase, C4H)和4-香豆酰CoA连接酶(4-coumarate--CoA ligase, 4CL)的催化下生成4-香豆酰CoA；第二阶段是4-香豆酰CoA和丙二酰CoA在查尔酮合成酶(chalcone synthase; CHS)、查尔酮异构酶(chalcone isomerase; CHI)、黄酰酮-3-羟化酶(flavanone-3-hydroxylase, F3H)的作用下生成黄酰酮和二羟黄酮醇，这个阶段也是花青素合成代谢的关键阶段；第三阶段是二羟黄酮醇在二羟黄酮醇还原酶(dihydroflavonol 4-reductase; DFR)的催化下生成无色花青素，再经花青素合酶(anthocyanin synthetase; ANS)和类黄酮-3-葡糖基转移酶(flavonoid-3-O-glucosyltransferase; 3GT)的催化生成有色花青素(Holton and Cornish, 1995; Ferrer et al., 2008)。此外，光照、温度、干旱等环境因子以及蔗糖、茉莉酸、乙烯等化合物的水平都会影响植物花青素的合成(Christie and Jenkins, 1996; Zhang et al., 2002; El-Kereamy et al., 2003; Hara et al., 2003; Mori et al., 2007; Sperdouli and Moustakas, 2012)。

小桐子(Jatropha curcas L.)属于多年生大戟科木本油料作物，其种子油具有很高的商业价值，可用于生产生物柴油(Pandey et al., 2012)和生物航空燃油(Du et al., 2019)。同时小桐子高效的遗传转化体系为改良其生物学性状奠定了坚实的基础(Pan et al., 2010; Fu et al., 2015)。但是目前小桐子功能基因的研究主要集中于筛选影响其与分枝、开花诱导和花器官发育以及种子油含量相关的基因(Li et al., 2014; Kim et al., 2014; Ni et al., 2015; Li et al., 2017; Ni et al., 2017; Khan et al., 2018; Govender et al., 2019)，对调控其花青素生物合成基因的研究较少。目前仅发现超量表达小桐子六磷酸海藻糖合成酶基因JcTPS1的拟南芥叶片中花青素含量增加(赵美丽等, 2018, 分子植物育种, 16(1): 255-261)。花青素可以保护植物免受各种生物和非生物胁迫(Zhang et al., 2013; Ahmed et al., 2014)。野生小桐子幼叶花青素含量明显高于成熟叶片，研究表明含量较高的花青素对小桐子幼叶的光系统I和光系统II具有保护作用(Ranjan et al., 2014)。茉莉酸可诱导小桐子叶片花青素的合成，从而增加植株的抗氧化能力(Lucho-Constantino et al., 2017)。

SEUSS (SEU)是动物LDB (LIM-domain-binding)蛋白的同源蛋白，由两个富含谷氨酰胺的结构域和一个高度保守的结构域组成，这个高度保守的结构域和动物的LIM结合结构域有很高的序列相似性(Franks et al., 2002)。SEU在拟南芥的幼苗、叶片和花等多个组织部位均有表达，参与了一系列生长发育过程(Franks et al., 2002; Bao et al., 2010)。主要参与调控拟南芥根、花器官以及胚珠和胚胎的发育等(Sridhar et al., 2006; Azhakanandam et al., 2008; Wynn et al., 2014; Gong et al., 2016)。本研究克隆了小桐子SEUSS (JcSEUSS1)的cDNA序列，对其表达模式进行分析，并通过在小桐子中超量表达该基因，初步研究其对小桐子叶中的花青素合成的影响。

1结果和分析

1.1 JcSEUSS的克隆和序列分析

以拟南芥SEU (NP_175051)蛋白序列作为参考，在小桐子基因组数据库(http://www.kazusa.or.jp/jatropha/)中进行同源序列比对，找到两个与拟南芥SEU有高相似性的序列，其编码的蛋白分别命名为JcSEUSS1 (XP_012066097)和JcSEUSS2 (XP_012066572)，两者的序列同一性百分比为58.44％。其中JcSEUSS1 cDNA(XM_012210707)序列长度为3 842 bp，CDS序列长度为2 748 bp，编码915个氨基酸。采用DNAMAN软件对小桐子JcSEUSS1氨基酸序列与其他几个物种的SEU蛋白序列进行比对，发现其与其它几个物种均具有高度保守的LIM结合结构域(图1)，因此可以推断小桐子JcSEUSS1属于LIM结构域蛋白。随后利用MEGA 10.0软件对JcSEUSS1与其他物种的同源蛋白进行系统进化树分析。结果显示小桐子(JcSEUSS1)与蓖麻(RcSEUSS)、木薯(MeSEUSS)和毛果杨(PtSEUSS)亲缘关系较近(图2)。

1.2小桐子JcSEUSS1的组织特异性表达模式

分别选取成年小桐子的根、茎、幼叶、成熟叶、花序、雌花、雄花和果实8个组织部位，利用实时荧光定量PCR对小桐子JcSEUSS1基因进行组织特异性表达分析。结果显示：JcSEUSS1在小桐子的各个组织部位均有表达(图3)。在根、幼叶和花序中的相对表达量较高，其中根中的相对表达量最高，且叶片中的表达量明显高于雌雄花。

1.3超量表达JcSEUSS1促进小桐子叶中的花青素的积累

为了探究JcSEUSS1基因对小桐子生长发育的影响，本研究构建了35S:JcSEUSS1的超量表达载体(图4A)，并通过农杆菌介导法转化小桐子。获得14株独立的转基因植株(图4B)，随机选取L3、L4和L7 3株T0代转基因株系进行表型分析。qRT-PCR检测发现(图4C)：这3株转基因植株叶柄中JcSEUSS1基因的表达量均明显上调；说明L3、L4和L7确实为阳性植株。其中L4表达量最高，表达量比野生型提高40倍，L3表达量最低，表达量比野生型提高约3倍。

随后观察比较T0代转基因株系表型，结果显示：转基因植株叶片和叶柄均明显变红(图5A~图5D)。对幼叶和成熟叶分别进行观察和分析后发现：幼叶叶片颜色变深，明显红于野生型，此外，不同于野生型嫩绿色的叶脉和叶柄，转基因植株的幼叶叶脉和叶柄呈现深红色(图5E)。花青素提取和测量结果显示(图5F)：转基因植株幼叶叶片和叶柄的花青素含量均明显高于野生型。3个转基因植株幼叶花青素含量较野生型均提高2倍左右，而叶柄花青素含量提高更明显，转基因植株L3提高约9倍，L4提高约15倍，L7提高约13倍。说明超量表达JcSEUSS1促进小桐子幼叶花青素的合成。

比较转基因植株成熟叶形态，发现野生型和不同转基因植株的成熟叶叶片均呈现绿色，但是转基因株系叶脉和叶柄明显呈红色(图5G)。花青素含量测定后发现(图5H)：转基因株系成熟叶叶片和叶柄花青素含量明显高于野生型，其中叶柄花青素提高更明显。L3转基因植株叶片花青素含量提高不显著，L4和L7较于野生型增加显著，而3个转基因植株叶柄花青素含量提高约10倍。结果显示无论成熟叶片还是幼嫩叶片和叶柄中，均是JcSEUSS1表达量最高的L4株系中的花青素含量最高，表达量最低的L3株系中的花青素含量最低。说明超量表达JcSEUSS1促进小桐子叶中花青素的积累。

1.4转基因小桐子幼叶叶柄中花青素合成相关基因表达量上调

对超量表达JcSEUSS1转基因小桐子幼叶叶柄中编码花青素合成关键酶基因JcCHS和JcCHI的相对表达量进行检测。结果显示：野生型小桐子幼叶叶柄花青素含量较低，这两个基因的表达量也很低；但是转基因植株中这两个基因的表达量都明显上调(图6)，且JcCHS和JcCHI的相对表达量趋势与JcSEUSS1一致。说明超量表达JcSEUSS1基因可能通过促进小桐子中花青素合成相关基因的表达，从而促进小桐子叶中花青素的积累。

2讨论

SEU基因在拟南芥中的研究较为广泛，拟南芥SEU基因在幼苗、叶、花序芽、花等多个组织部位均有表达，所以SEU基因调控拟南芥营养生长和生殖生长的多个生长发育阶段(Franks et al., 2002; Bao et al., 2010)。本研究发现：JcSEUSS1在小桐子中也是一个类似组成型表达的基因，且调控小桐子不同组织部位，包括叶片、雄花、果实和种子的生长发育过程。说明与其他物种比较，JcSEUSS1不仅在蛋白序列上高度保守，而且基因功能也很保守。在拟南芥中，SEU基因在花中的表达量明显高于叶片中的表达量，所以SEU主要调控了拟南芥花的发育(Franks et al., 2002)。但是在小桐子中，JcSEUSS1在叶片中的表达量明显高于雌雄花，所以SEU基因的功能在小桐子和拟南芥中可能存在一定差异，JcSEUSS1对小桐子叶发育的影响可能强于其对花发育的影响。

本研究发现，超量表达JcSEUSS1基因会促进小桐子叶中花青素的积累，目前其他物种中还没有SEU基因调控植物花青素合成的相关报道。拟南芥中虽然没有报道过SEU调控花青素合成的功能，但是发现SEU与花青素合成关键酶之间存在调控关系：无论在暗培养还是持续光照条件下，相较于野生型，其突变体seu-6中编码类黄酮合成的关键酶基因CHS的表达量都明显上调(Huai et al., 2018)。SEU可与PIF4相互作用，二者均负调控拟南芥的光形态发生，且正调控温度介导的下胚轴伸长 (Huai et al., 2018)；而PIF4属于helix-loop-helix (bHLH)转录因子家族成员，是红光诱导花青素积累的负调控因子(Liu et al., 2015)。在本研究中，我们发现超量表达JcSEUSS1会导致JcCHS和JcCHI基因的表达量明显上调(图6)，qPCR检测结果显示转基因植株中JcSEUSS1表达量明显上升并未发生基因沉默(图4)。这与拟南芥中SEU影响CHS的表达结果(Huai et al., 2018)相反，说明小桐子JcSEUSS1基因的功能与模式植物拟南芥中的同源基因的功能可能存在差异。该差异有可能与本研究中的转基因小桐子种植于光照较强、温度较高的热带地区相关，因为已有研究表明光照强度和光质对植物花青素积累存在重要调节作用(Dong et al., 1998)。植物CHS基因编码查尔酮合成酶，是类黄酮合成过程中的第一个关键酶(Tropf et al., 1995)。CHS基因的表达需要UV-B和UV-A/蓝光的诱导(Christie and Jenkins, 1996)。拟南芥中，超量表达CHS基因可增加叶片对强光的耐受能力(Zhang et al., 2018)。CHI编码查尔酮异构酶，其功能主要是将CHS催化产生的黄色查尔酮转变为无色的黄烷酮，CHI也是花青素合成途径的关键酶(Burbulis and Winkel-Shirley, 1999)。其表达也需要光的诱导(Song et al., 1998)。由此可见，CHS和CHI均为植物光诱导花青素合成代谢通路所必须的酶，而在小桐子JcSEUSS1基因超量表达植株中JcCHS和JcCHI的表达量都明显上调，说明JcSEUSS1可能参与了小桐子光诱导花青素合成代谢通路，通过调控JcCHS和JcCHI基因的表达来促进小桐子叶中的花青素含量的积累。

小桐子主要在光照和紫外线都较强的热带和亚热带地区种植，而花青素具有光保护作用(Lucho-Constantino et al., 2017; 庞丹丹等, 2018)；本研究中获得的超量表达JcSEUSS1转基因植株叶片和叶柄中花青素含量提高了2~10倍(图5F, 图5H)，因而可尝试将这些转基因材料用于培育抗逆的小桐子优良品种。

3材料与方法

3.1植物材料和种植

本实验所用小桐子，幼苗培养于云南省昆明市西双版纳热带植物园昆明分部组培室(25°05′N, 102°69′E)，温度为(26±2)°C，光照为14 h/d。成年植株种植于云南省西双版纳自治州勐腊县勐仑镇中国科学院西双版纳热带植物园(21°41′N, 101°25′E)，年平均气温21.4°C，年降雨量1 556 mm。

3.2小桐子JcSEUSS1全长cDNA的克隆

利用硅胶吸附法提取两年生小桐子的根、茎、幼叶(自茎尖展叶开始的第二片幼叶)、成熟叶(自茎尖展叶开始的第八片叶)、花序、雌花、雄花和生长40 d的果实的总RNA (Ding et al., 2008)。并用琼脂糖凝胶电泳和NanoDrop 2000分光光度计对小桐子RNA的质量和浓度进行检测。再参考TAKARA (大连, 中国) PrimeScript ^TH Reagent Kit with gDNA Eraser说明书将RNA反转录为cDNA。以小桐子基因组数据库(http://www.kazusa.or.jp/jatropha/)得到的JcSEUSS1核苷酸序列为模板，使用Primer5设计引物XC772和XC773 (表1)，并扩增JcSEUSS1的全长cDNA。

3.3实时荧光定量PCR

首先在NCBI网站(https://www.ncbi.nlm.nih.gov/)上设计基因的qRT-PCR引物，再以cDNA为模板，使用Roche的LightCycler 480 SYBR Green I Master试剂盒进行qRT-PCR实验，使用LightCycler 480软件对导出数据进行分析，使用Sigmablot软件作图。以小桐子JcACT1基因作为内参，试验采用3次生物学重复。

3.4 35S:JcSEUSS1质粒构建和转化小桐子

将测序正确无碱基突变的JcSEUSS1全长cDNA序列用BamHI和SalI限制性内切酶切割以后连接到含有CaMV 35S启动子和35S增强子的pOCA30载体上，构建超量表达载体35S:JcSEUSS1，利用农杆菌介导转化法转化小桐子。本试验以小桐子种子的子叶作为转化外植体进行农杆菌侵染，再利用卡那霉素抗性和PCR扩增筛选阳性植株(Pan et al., 2010; Fu et al., 2015)。

3.5小桐子花青素的提取

本试验参考修改后的Gou等(2011)的花青素提取方法，取1 g一年生的小桐子组织样品，液氮速冻碾磨后，加入4 mL的花青素提取液(5％的盐酸和80％的甲醇)，混匀后置于4°C过夜，14 000 r/min离心20 min，取上清。使用NanoDrop2000分光光度计分别于530 nm、620 nm、650 nm 3个波长下测量OD值。

花青素含量(nmol/g)=OD_λ/Ɛ×v/m×1 000 000

花青素的光密度值OD_λ=(OD₅₃₀-OD₆₂₀)-0.1 (OD₆₅₀-OD₆₂₀)

OD_λ：花青素的光密度值，Ɛ：花青素的摩尔消光系数4.62×10⁶，v：萃取液的总体积(mL)，m：样品质量(g)。

作者贡献

王静娴是本实验研究的执行者，完成了实验数据分析以及初稿的写作；明新、陶彦彬、唐明勇和徐增富在实验过程中提供了帮助并参与了论文的修改；唐明勇和徐增富是项目的构思者及负责人，指导实验设计和数据分析。全体作者都阅读并同意最终的文本。

致谢

本研究由云南省科技厅科技计划面上项目(2018FB060)、国家自然科学基金委青年科学家基金项目(31700273)和中国科学院项目(kfj-brsn-2018-6-008, 2017XTBG-T02)共同资助。

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