CN111855920A - 一种鉴定棉花受碱胁迫程度的方法 - Google Patents
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Abstract
本发明提供一种鉴定棉花受碱胁迫程度的方法，包括：1)以沙培法种植棉花幼苗至三叶期，将三叶期的棉花幼苗洗净，分别移栽到含有水和25、50、75mmol·L–1Na2CO3溶液的锥形瓶中培养24h后，观察叶色变化；采集幼苗叶片，测定不同碱浓度下花青素的总含量；绘制碱浓度‑花青素总含量的标准曲线；采集待鉴定棉花三叶期幼苗叶片，观察叶片颜色，比对，预估其受碱胁迫程度，然后提取花青素测定花青素的总含量；将得到的花青素的总含量代入标准曲线中，计算得到对应的碱浓度，即，棉花受碱胁迫程度。本发明利用叶片中花青素变化来判断棉花受到碱胁迫的程度，可以为快速筛选耐碱棉花品种提供依据。
Description
技术领域
本发明涉及生物技术领域，具体涉及一种鉴定棉花受碱胁迫程度的方法。
背景技术
受人类活动的影响，土壤盐碱化现象日趋加重，碱胁迫作为影响植物生长发育的重要非生物胁迫之一，严重制约了制约农作物产量和持续性生态环境体系建设的重要因素(Zhu and Li,2013)。碱胁迫对植物造成的危害不仅包括高盐离子浓度引发的氧化胁迫和渗透胁迫，还有高pH毒害作用(Shi et al.,2005)。目前全国的盐碱地多达9,913万公顷，大多数研究集中于耐盐机制的研究，而忽略了碱土中的Na2CO3、NaHCO3对植物造成的损伤(Shiet al.,2005)。棉花在我国不仅是一种重要的经济作物，也是适应盐碱环境的先锋植物，因此深入探究棉花耐碱机制对于有效地提高棉花耐碱性、培育耐碱品种具有重要的意义。
植物在长期进化和发育过程中形成了多种次生代谢物帮助其抵御逆境胁迫，适应环境变化(Martens et al.,2010)。花青素就是植物应对环境变化产生的一种黄酮类次生代谢产物。花青素具备极强的抗氧化性和清除自由基能力，植物在应对逆境胁迫时能通过合成花青素清除体内过多的活性氧，从而有效抵御活性氧胁迫。研究发现，臭氧能够诱导帚石楠(Calluna vulgaris)幼苗产生大量的花青素，表明花青素在降低活性氧胁迫中起作用(Foot et al.,1996)，另有研究表明，随着贮藏时间的增加，笃斯越桔中花青素含量降低，对活性氧清除能力也显著降低(张晓松和李冬梅,2008)。因此花青素对于植物响应逆境胁迫起着至关重要的作用。
此外，花青素作为一种常见的次生代谢产物，不仅广泛的存在于植物的叶片、花朵和果实中，影响着植物花朵和果实的颜色，而且在提高植物的抗氧化性、抵御逆境胁迫方面起到重要作用。有研究表明，在棉花受到虫害时叶片出现了明显的红色花青素积累，从而把花青素的积累量当作受害程度的指示剂(Kangatharalingam et al.,2002)。
探索一种快速指示棉花受到碱胁迫程度的方法是亟待解决的重要问题。
发明内容
本发明的目的是提供一种鉴定棉花受碱胁迫程度的方法。
本发明所提供的鉴定棉花受碱胁迫程度的方法，包括如下步骤：
1)以沙培法种植棉花幼苗至三叶期，将三叶期的棉花幼苗洗净，分别移栽到含有水和25、50、75mmol·L–1Na2CO3溶液的锥形瓶中培养24h后，观察叶色变化；
2)采集步骤1)中棉花幼苗叶片，从叶片中提取花青素，测定不同碱浓度下花青素的总含量；
3)以Na2CO3溶液的浓度为横坐标，以测得的棉花叶片中花青素的总含量为纵坐标，绘制碱浓度-花青素总含量的标准曲线；
4)采集待鉴定棉花三叶期幼苗叶片，观察叶片颜色，与步骤1)中的不同浓度碱处理后的叶片颜色进行对比，预估其受碱胁迫程度，然后参照步骤2)的操作从叶片中提取花青素，测定花青素的总含量；将得到的花青素的总含量代入步骤3)中的标准曲线中，计算得到对应的碱浓度，即，棉花受碱胁迫程度。
上述方法步骤2)的操作为：采集步骤1)中棉花幼苗叶片，剪碎，加入含有体积分数为1％的HCl甲醇溶液，于4℃黑暗条件下放置24h至叶片完全发白，用含有1％HCl的甲醇溶液作为空白对照，测定实验组溶液在530nm和657nm处的吸光值，得到棉花叶片中花青素的含量；
其中，所述棉花叶片与含有1％HCl的甲醇溶液的配比可为：0.1g：0.6mL。
上述方法还可进一步包括采用液质联用定性分析碱胁迫下花青素成分的变化，结合生物信息学分析确定影响叶色变化的主要成分。
本发明利用叶片中花青素变化来判断棉花受到碱胁迫的程度，可以为快速筛选耐碱棉花品种提供依据。
附图说明
图1表示本发明实施例中0、25、50、75mM Na2CO3溶液处理24h后棉花叶片的颜色变化，即，花青素积累的表型图，从左到右依次是0、25、50、75mM Na2CO3溶液处理24h后棉花叶片的颜色变化。
图2为不同浓度碱胁迫处理后叶片中花青素含量变化。
图3为碱浓度-花青素总含量的标准曲线。
图4为液质联用分析导致叶色变化的主要成分。
具体实施方式
下面通过具体实施例对本发明进行说明，但本发明并不局限于此。
下述实施例中所使用的实验方法如无特殊说明，均为常规方法；下述实施例中所用的试剂、材料等，如无特殊说明，均可从商业途径得到。
实施例
1、中9807种子脱绒后，浸泡在体积分数为75％酒精里消毒10min。采用沙培法种植，幼苗的生长条件为14h光照/10h黑暗，25℃。将三叶期的棉花幼苗仔细洗净，分别移栽到含有水和25、50、75mmol·L–1Na2CO3溶液的锥形瓶中培养24h后，观察叶色变化(见图1)并取样用于后续实验。
由图1可以看出：随着碱胁迫浓度逐渐提高，花青素的积累量逐渐增加；
2、取0.1g新鲜的棉花叶片剪碎后放于离心管中，加入0.6mL含有体积分数1％HCl的甲醇溶液，于4℃黑暗条件下放置24h至叶片完全发白；用含有1％HCl的甲醇溶液作为空白对照，在紫外可见分光光度计的530nm和657nm处测定所得溶液的吸光值，所有样品均重复3次，即可测定棉花叶片中花青素的含量(见图2，纵坐标单位为mg/g)；
3、以Na2CO3溶液的浓度为横坐标，以测得的棉花叶片中花青素的总含量为纵坐标，绘制碱浓度-花青素总含量的标准曲线(见图3)，由图3可以看出，在Na2CO3浓度在0-75mmol·L–1范围内，花青素含量与碱(碳酸钠)浓度呈线性关系，且，R2＝0.9779；
4)采集待鉴定棉花三叶期幼苗叶片，观察叶片颜色，与步骤1)中的不同浓度碱处理后的叶片颜色进行对比，预估其受碱胁迫程度，然后参照步骤2)的操作从叶片中提取花青素，测定花青素的总含量；将得到的花青素的总含量代入步骤3)中的标准曲线中，计算得到对应的碱浓度，即，棉花受碱胁迫程度。
5)液质联用定性分析碱胁迫下花青素成分的变化，结合生物信息学分析确定影响叶色变化的主要成分，
表1标准品与试剂
Table1 Standards and reagents
(2)样品提取流程
①取样时，使用液氮快速冷冻生物样品，放于超低温冰箱备用；
②将样品放在30Hz的研磨仪中，充分研磨至样品呈粉末状；
③称取100mg粉末状样品，溶解于70％的甲醇水溶液中；
④样品置于4℃冰箱中过夜提取；
⑤10,000g，离心10分钟后，吸取上清液；
⑥用直径为0.22μm的微孔滤膜过滤样品，并保存于进样瓶中，用于后续实验。
(3)色谱质谱采集条件
数据采集仪器系统主要由两部分组成，分别是超高效液相色谱仪(UltraPerformance Liquid Chromatography)和串联质谱仪(Tandem mass spectrometry,MS/MS)。
液相条件如下：
①色谱柱的型号为Waters ACQUITY UPLC HSS T3 C18；
②流动相中的流动相A为加入了0.04％乙酸的乙腈；流动相B为加入了0.04％乙酸的超纯水；
③洗脱程序设定：0min水/乙腈为90％，12.0min为20％，12.1min为90％，15min为90％；
④流速设定为0.4mL·min–1；柱温40℃；进样量5μL。
表2质谱检测参数
Table2 The parameters of mass spectrometrydetected
表3、50mM碱胁迫下差异表达代谢物
Table3 Differential expression of metabolites under alkaline stress
由表3可以看出，碱胁迫下花青素总含量的变化主要体现在原花青素A1的大幅度上调，以及牵牛花色素3-O-葡萄糖的上调。
Claims (5)
1.一种鉴定棉花受碱胁迫程度的方法，包括如下步骤：
1)以沙培法种植棉花幼苗至三叶期，将三叶期的棉花幼苗洗净，分别移栽到含有水和25、50、75mmol·L–1Na2CO3溶液的锥形瓶中培养24h后，观察叶色变化；
2)采集步骤1)中棉花幼苗叶片，从叶片中提取花青素，测定不同碱浓度下花青素的总含量；
3)以Na2CO3溶液的浓度为横坐标，以测得的棉花叶片中花青素的总含量为纵坐标，绘制碱浓度-花青素总含量的标准曲线；
4)采集待鉴定棉花三叶期幼苗叶片，观察叶片颜色，与步骤1)中的不同浓度碱处理后的叶片颜色进行对比，预估其受碱胁迫程度，然后参照步骤2)的操作从叶片中提取花青素，测定花青素的总含量；将得到的花青素的总含量代入步骤3)中的标准曲线中，计算得到对应的碱浓度，即，棉花受碱胁迫程度。
2.根据权利要求1所述的方法，其特征在于：步骤2)的操作为：采集步骤1)中棉花幼苗叶片，剪碎，加入HCl体积分数为1％的甲醇溶液，于4℃黑暗条件下放置24h至叶片完全发白，用含有1％HCl的甲醇溶液作为空白对照，测定所得溶液在530nm和657nm处的吸光值，得到棉花叶片中花青素的含量；
其中，所述棉花叶片与含有1％HCl的甲醇溶液的配比为：0.1g：0.6mL。
3.根据权利要求1或2所述的方法，其特征在于：所述方法进一步包括采用液质联用定性分析碱胁迫下花青素成分的变化，结合生物信息学分析确定影响叶色变化的主要成分。
4.权利要求1-3中任一项所述方法在选育耐碱胁迫棉花品种中的应用。
5.花青素在选育耐碱胁迫棉花品种中的应用。
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