CN110760512A - 一种抑制蓝莓多酚氧化酶活性的反义核酸及其应用 - Google Patents
一种抑制蓝莓多酚氧化酶活性的反义核酸及其应用 Download PDFInfo
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Abstract
一种抑制蓝莓多酚氧化酶活性的反义核酸，所述反义核酸为正义链和反义链两条完全互补配对单链组成的双链，所述正义链和所述反义链均由483个碱基组成。以及上述抑制蓝莓多酚氧化酶活性的反义核酸在蓝莓组培苗过程中的应用。本发明解决了现有的蓝莓组培苗制作过程中褐化物质的产生和毒害的问题，显著抑制蓝莓苗组培快繁过程中的褐化现象。
Description
技术领域
本发明涉及反义核酸领域，特别是涉及一种抑制蓝莓多酚氧化酶活性的反义核酸及其应用。
背景技术
蓝莓是一种小浆果，果实呈蓝色、果肉细腻、种子极小，具有“浆果之王”和“水果皇后”的美誉。蓝莓果实平均重0.5～2.5g，最大重5g，可食率为100％，甜酸适口，且具有香爽宜人的香气，为鲜食佳品。蓝莓不仅富含常规营养成分，如蛋白质、维生素、矿物质及微量元素等，而且还含有极为丰富的花青素、黄酮类和多糖类化合物，具有缓解视力疲劳，促进视网膜细胞中的视紫质再生，预防近视，改善和增进视力等功效。
除缓解视力疲劳外，蓝莓还具有诸多保健功能，如预防心脑血管疾病、防癌、提高免疫力、缓解咽喉疼痛及腹泻等等，所以，对蓝莓的利用和衍生品开发越来越受到重视。
鉴于蓝莓的广阔市场前景，对蓝莓苗木的研发也越来越受到重视。其中快速脱毒制备蓝莓组培苗成为最佳蓝莓苗木繁殖手段。然而在组培快繁过程中，因为外植体创面的细胞受伤害造成细胞内酚类化合物外泌，在多酚氧化酶的作用下形成褐色的醌类物质，在培养基中积聚并逐渐扩散，抑制其它酶的活性，毒害整个外植体，进而造成组培苗死亡，不利于蓝莓苗的快速繁殖，是蓝莓组培苗成功与否的决定性因素之一。
因此，基于蓝莓苗组培褐化问题，需要一种有效的技术手段抑制蓝莓多酚氧化酶活性，解决以上问题。
发明内容
本发明的目的在于提供一种抑制蓝莓多酚氧化酶活性的反义核酸及其应用，解决了现有的蓝莓组培苗制作过程中褐化物质的产生和毒害的问题，显著抑制蓝莓苗组培快繁过程中的褐化。
为实现上述目的，本发明是通过以下技术方案来实现的：
一种抑制蓝莓多酚氧化酶活性的反义核酸，所述反义核酸为正义链和反义链两条完全互补配对单链组成的双链，所述正义链和所述反义链均由483个碱基组成，所述正义链序列为：5’
aaaguagcgaagaagcugggaguugcacacgcagcuugugcgucguccagcaaggguuguggcaggcacugaguuucccgauaagucuggggucgaagauaagcacgguggugaagagaccgaagcagaagaagaggagcaagaaggccaaggaggaugaggaggagauauuggugauugagggaaucgaguuugacagggacguggcugugaaguuugauguguaugugaaugacgucgaugacuugccgagugggccugacaagacggaguuugccggaagcuuuguaagugugccgcacagccacaagcacaagaagaagaugaacacuauuuugagguuaggguugacagauuuguuggaggaaauugaggcggaggacgaugacagcgugguggugacuuuggugcccaaguucggcgcugucaagauuggugguaucaagauugaauuugcuuuuuaguuaguuaugucaacccagg 3’，
所述反义链序列为：5’
ccuggguugacauaacuaacuaaaaagcaaauucaaucuugauaccaccaaucuugacagcgccgaacuugggcaccaaagucaccaccacgcugucaucguccuccgccucaauuuccuccaacaaaucugucaacccuaaccucaaaauaguguucaucuucuucuugugcuuguggcugugcggcacacuuacaaagcuuccggcaaacuccgucuugucaggcccacucggcaagucaucgacgucauucacauacacaucaaacuucacagccacgucccugucaaacucgauucccucaaucaccaauaucuccuccucauccuccuuggccuucuugcuccucuucuucugcuucggucucuucaccaccgugcuuaucuucgaccccagacuuaucgggaaacucagugccugccacaacccuugcuggacgacgcacaagcugcgugugcaacucccagcuucuucgcuacuuu 3’。
以及上述抑制蓝莓多酚氧化酶活性的反义核酸在蓝莓组培苗过程中的应用。
本发明反义核酸的制备过程，包括以下步骤：
1.根据基因序列设计含有但不限于T7启动子的上、下游引物，也可以上、下游引物含有不同的启动子，如T7和T3。启动子的作用在于体外转录时，根据基因的转录原理，用于启动对应的目的基因转录成mRNA。引物设计完成并检查无误后合成。
2.获得含有目标基因的质粒。
3.以含有目标基因的质粒为模板，通过常规PCR方法扩增目标基因片段。
4.电泳检测PCR产物，并纯化回收PCR产物。
5.以PCR产物为模板进行体外转录获得转录产物，如果是相同的启动子则一次转录即可。如果是不同启动子，需要分别转录，然后将两次转录产物等比例混合退火形成双链反义核酸。
6.转录产物经电泳检测，并纯化回收，测定浓度后，备用。
将本发明应用于蓝莓组培苗过程，制备蓝莓组培苗快繁的固体培养基，包括以下步骤：
1.配置基本培养基，所用的培养基为本领域通用培养基，本实验选用WPM培养基。
2.将适量的琼脂加入WPM培养基中。
3.向步骤2的培养基中加入本发明反义核酸。混合搅拌均匀后分装，每瓶20-30ml，灭菌后备用。
为确定最佳配制比例，将本发明反义核酸按不同的比例加入蓝莓组培苗快繁的培养基中，溶解混匀后分装，用于制作蓝莓组培苗快繁的固体培养基，并观察结果。
通过实验结果发现，每升WPM培养基中，加入至少1mmol本发明反义核酸，在蓝莓组培苗快速繁殖培养过程中，瓶苗不发生褐化，成活率显著提高。
本发明的有益效果，本发明的反义核酸具有抑制多酚氧化酶活性的功能，能够减轻多酚氧化酶造成的醌类褐化物在培养基中的分散作用以及对蓝莓组培苗的毒害。使用本发明反义核酸培育蓝莓组培苗，较传统技术手段而言，其对于多酚氧化酶活性抑制效果好，且表现稳定，瓶苗不发生褐化，成活率显著提高。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将对本发明实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用组分或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
下面对本发明作进一步说明如下：
实施例一
一种抑制蓝莓多酚氧化酶活性的反义核酸，所述反义核酸为正义链和反义链两条完全互补配对单链组成的双链，所述正义链和所述反义链均由483个碱基组成，所述正义链序列为：5’
aaaguagcgaagaagcugggaguugcacacgcagcuugugcgucguccagcaaggguuguggcaggcacugaguuucccgauaagucuggggucgaagauaagcacgguggugaagagaccgaagcagaagaagaggagcaagaaggccaaggaggaugaggaggagauauuggugauugagggaaucgaguuugacagggacguggcugugaaguuugauguguaugugaaugacgucgaugacuugccgagugggccugacaagacggaguuugccggaagcuuuguaagugugccgcacagccacaagcacaagaagaagaugaacacuauuuugagguuaggguugacagauuuguuggaggaaauugaggcggaggacgaugacagcgugguggugacuuuggugcccaaguucggcgcugucaagauuggugguaucaagauugaauuugcuuuuuaguuaguuaugucaacccagg 3’，
所述反义链序列为：5’
ccuggguugacauaacuaacuaaaaagcaaauucaaucuugauaccaccaaucuugacagcgccgaacuugggcaccaaagucaccaccacgcugucaucguccuccgccucaauuuccuccaacaaaucugucaacccuaaccucaaaauaguguucaucuucuucuugugcuuguggcugugcggcacacuuacaaagc uuccggcaaacuccgucuugucaggcccacucggcaagucaucgacgucauucacauacacaucaaacuucacagccacgucccugucaaacucgauucccucaaucaccaauaucuccuccucauccuccuuggccuucuugcuccucuucuucugcuucggucucuucaccaccgugcuuaucuucgaccccagacuuaucgggaaacucagugccugccacaacccuugcuggacgacgcacaagcugcgugugcaacucccagcuucuucgcuacuuu 3’。
制备本发明反义核酸，首先通过PCR扩增获得具有T7启动子的模板序列，然后进行体外转录。按上述过程获得反义核酸后，测定浓度并根据要求保存备用。然后制备反义核酸为1mmol/L的蓝莓组培苗快繁的固体培养基，标记为培养基一。
实施例二
一种抑制蓝莓多酚氧化酶活性的反义核酸，所述反义核酸为正义链和反义链两条完全互补配对单链组成的双链，所述正义链和所述反义链均由483个碱基组成，所述正义链序列为：5’
aaaguagcgaagaagcugggaguugcacacgcagcuugugcgucguccagcaaggguuguggcaggcacugaguuucccgauaagucuggggucgaagauaagcacgguggugaagagaccgaagcagaagaagaggagcaagaaggccaaggaggaugaggaggagauauuggugauugagggaaucgaguuugacagggacguggcugugaaguuugauguguaugugaaugacgucgaugacuugccgagugggccugacaagacggaguuugccggaagcuuuguaagugugccgcacagccacaagcacaagaagaagaugaacacuauuuugagguuaggguugacagauuuguuggaggaaauugaggcggaggacgaugacagcgugguggugacuuuggugcccaaguucggcgcugucaagauuggugguaucaagauugaauuugcuuuuuaguuaguuaugucaacccagg 3’，
所述反义链序列为：5’
ccuggguugacauaacuaacuaaaaagcaaauucaaucuugauaccaccaaucuugacagcgccgaacuugggcaccaaagucaccaccacgcugucaucguccuccgccucaauuuccuccaacaaaucugucaacccuaaccucaaaauaguguucaucuucuucuugugcuuguggcugugcggcacacuuacaaagcuuccggcaaacuccgucuugucaggcccacucggcaagucaucgacgucauucacauacacaucaaacuucacagccacgucccugucaaacucgauucccucaaucaccaauaucuccuccucauccuccuuggccu ucuugcuccucuucuucugcuucggucucuucaccaccgugcuuaucuucgaccccagacuuaucgggaaacucagugccugccacaacccuugcuggacgacgcacaagcugcgugugcaacucccagcuucuucgcuacuuu 3’。
制备本发明反义核酸，首先通过PCR扩增获得具有T7启动子的模板序列，然后进行体外转录。按上述过程获得反义核酸后，测定浓度并根据要求保存备用。然后制备反义核酸为1.5mmol/L的蓝莓组培苗快繁的固体培养基，标记为培养基二。
实施例三
一种抑制蓝莓多酚氧化酶活性的反义核酸，所述反义核酸为正义链和反义链两条完全互补配对单链组成的双链，所述正义链和所述反义链均由483个碱基组成，所述正义链序列为：5’
aaaguagcgaagaagcugggaguugcacacgcagcuugugcgucguccagcaaggguuguggcaggcacugaguuucccgauaagucuggggucgaagauaagcacgguggugaagagaccgaagcagaagaagaggagcaagaaggccaaggaggaugaggaggagauauuggugauugagggaaucgaguuugacagggacguggcugugaaguuugauguguaugugaaugacgucgaugacuugccgagugggccugacaagacggaguuugccggaagcuuuguaagugugccgcacagccacaagcacaagaagaagaugaacacuauuuugagguuaggguugacagauuuguuggaggaaauugaggcggaggacgaugacagcgugguggugacuuuggugcccaaguucggcgcugucaagauuggugguaucaagauugaauuugcuuuuuaguuaguuaugucaacccagg 3’，
所述反义链序列为：5’
ccuggguugacauaacuaacuaaaaagcaaauucaaucuugauaccaccaaucuugacagcgccgaacuugggcaccaaagucaccaccacgcugucaucguccuccgccucaauuuccuccaacaaaucugucaacccuaaccucaaaauaguguucaucuucuucuugugcuuguggcugugcggcacacuuacaaagcuuccggcaaacuccgucuugucaggcccacucggcaagucaucgacgucauucacauacacaucaaacuucacagccacgucccugucaaacucgauucccucaaucaccaauaucuccuccucauccuccuuggccuucuugcuccucuucuucugcuucggucucuucaccaccgugcuuaucuucgaccccagacuuaucgggaaacucagugccugccacaacccuugcuggacgacgcacaagcugcgugugcaacucccagcuucuuc gcuacuuu 3’。
制备本发明反义核酸，首先通过PCR扩增获得具有T7启动子的模板序列，然后进行体外转录。按上述过程获得反义核酸后，测定浓度并根据要求保存备用。然后制备反义核酸为2mmol/L的蓝莓组培苗快繁的固体培养基，标记为培养基三。
对比例一
一种抑制蓝莓多酚氧化酶活性的反义核酸，所述反义核酸为正义链和反义链两条完全互补配对单链组成的双链，所述正义链和所述反义链均由483个碱基组成，所述正义链序列为：5’
aaaguagcgaagaagcugggaguugcacacgcagcuugugcgucguccagcaaggguuguggcaggcacugaguuucccgauaagucuggggucgaagauaagcacgguggugaagagaccgaagcagaagaagaggagcaagaaggccaaggaggaugaggaggagauauuggugauugagggaaucgaguuugacagggacguggcugugaaguuugauguguaugugaaugacgucgaugacuugccgagugggccugacaagacggaguuugccggaagcuuuguaagugugccgcacagccacaagcacaagaagaagaugaacacuauuuugagguuaggguugacagauuuguuggaggaaauugaggcggaggacgaugacagcgugguggugacuuuggugcccaaguucggcgcugucaagauuggugguaucaagauugaauuugcuuuuuaguuaguuaugucaacccagg 3’，
所述反义链序列为：5’
ccuggguugacauaacuaacuaaaaagcaaauucaaucuugauaccaccaaucuugacagcgccgaacuugggcaccaaagucaccaccacgcugucaucguccuccgccucaauuuccuccaacaaaucugucaacccuaaccucaaaauaguguucaucuucuucuugugcuuguggcugugcggcacacuuacaaagcuuccggcaaacuccgucuugucaggcccacucggcaagucaucgacgucauucacauacacaucaaacuucacagccacgucccugucaaacucgauucccucaaucaccaauaucuccuccucauccuccuuggccuucuugcuccucuucuucugcuucggucucuucaccaccgugcuuaucuucgaccccagacuuaucgggaaacucagugccugccacaacccuugcuggacgacgcacaagcugcgugugcaacucccagcuucuucgcuacuuu 3’。
制备本发明反义核酸，首先通过PCR扩增获得具有T7启动子的模板序列，然后进行体外转录。按上述过程获得反义核酸后，测定浓度并根据要求保存备用。然后制备反义核酸为0.25mmol/L的蓝莓组培苗快繁的固体培养基，标记为培养基四。
对比例二
一种抑制蓝莓多酚氧化酶活性的反义核酸，所述反义核酸为正义链和反义链两条完全互补配对单链组成的双链，所述正义链和所述反义链均由483个碱基组成，所述正义链序列为：5’
aaaguagcgaagaagcugggaguugcacacgcagcuugugcgucguccagcaaggguuguggcaggcacugaguuucccgauaagucuggggucgaagauaagcacgguggugaagagaccgaagcagaagaagaggagcaagaaggccaaggaggaugaggaggagauauuggugauugagggaaucgaguuugacagggacguggcugugaaguuugauguguaugugaaugacgucgaugacuugccgagugggccugacaagacggaguuugccggaagcuuuguaagugugccgcacagccacaagcacaagaagaagaugaacacuauuuugagguuaggguugacagauuuguuggaggaaauugaggcggaggacgaugacagcgugguggugacuuuggugcccaaguucggcgcugucaagauuggugguaucaagauugaauuugcuuuuuaguuaguuaugucaacccagg 3’，
所述反义链序列为：5’
ccuggguugacauaacuaacuaaaaagcaaauucaaucuugauaccaccaaucuugacagcgccgaacuugggcaccaaagucaccaccacgcugucaucguccuccgccucaauuuccuccaacaaaucugucaacccuaaccucaaaauaguguucaucuucuucuugugcuuguggcugugcggcacacuuacaaagcuuccggcaaacuccgucuugucaggcccacucggcaagucaucgacgucauucacauacacaucaaacuucacagccacgucccugucaaacucgauucccucaaucaccaauaucuccuccucauccuccuuggccuucuugcuccucuucuucugcuucggucucuucaccaccgugcuuaucuucgaccccagacuuaucgggaaacucagugccugccacaacccuugcuggacgacgcacaagcugcgugugcaacucccagcuucuucgcuacuuu 3’。
制备本发明反义核酸，首先通过PCR扩增获得具有T7启动子的模板序列，然后进行体外转录。按上述过程获得反义核酸后，测定浓度并根据要求保存备用。然后制备反义核酸为0.5mmol/L的蓝莓组培苗快繁的固体培养基，标记为培养基五。
抑制蓝莓组培苗褐化实验
供试蓝莓品种：半高丛蓝莓品种“北陆”。
实验背景：无菌环境中，“北陆”蓝莓在现有的培育方式下，即不加入本发明的反义核酸来抑制褐化现象，其褐化率为15％，成活率为85％。
选取“北陆”蓝莓分成等量的五组进行实验，其培养基分别为培养基一、培养基二、培养基三、培养基四和培养基五。在无菌组培室内同时培育。培育180天后，观察实验结果，并记录，统计结果如表1所示。
表1抑制蓝莓组培苗褐化实验结果统计表
根据表1数据可知，本发明的反义核酸对蓝莓组培苗的褐化有抑制作用，并且培养基中的反义核酸浓度≥1mmol/L能够完全抑制蓝莓组培苗的褐化，其中反义核酸最佳浓度为1mmol/L。蓝莓组培苗的褐化被抑制，其成活率便明显提高，产量增加。本发明优异而稳定的抑制效果极大程度的优化了蓝莓组培苗快速繁殖培养过程。
尽管本发明的实施方案已公开如上，但其并不仅仅限于说明书和实施方式中所列运用。它完全可以被适用于各种适合本发明的领域。对于熟悉本领域的人员而言，可容易地实现另外的修改。因此在不背离权利要求及等同范围所限定的一般概念下，本发明并不限于特定的细节和这里示出与描述的表。
本专利申请核苷酸序列表
正义链序列为：5'
aaaguagcgaagaagcugggaguugcacacgcagcuugugcgucguccagcaaggguuguggcaggcacugaguuucccgauaagucuggggucgaagauaagcacgguggugaagagaccgaagcagaagaagaggagcaagaaggccaaggaggaugaggaggagauauuggugauugagggaaucgaguuugacagggacguggcugugaaguuugauguguaugugaaugacgucgaugacuugccgagugggccugacaagacggaguuugccggaagcuuuguaagugugccgcacagccacaagcacaagaagaagaugaacacuauuuugagguuaggguugacagauuuguuggaggaaauugaggcggaggacgaugacagcgugguggugacuuuggugcccaaguucggcgcugucaagauuggugguaucaagauugaauuugcuuuuuaguuaguuaugucaacccagg 3'；
反义链序列为：5'
ccuggguugacauaacuaacuaaaaagcaaauucaaucuugauaccaccaaucuugacagcgccgaacuugggcaccaaagucaccaccacgcugucaucguccuccgccucaauuuccuccaacaaaucugucaacccuaaccucaaaauaguguucaucuucuucuugugcuuguggcugugcggcacacuuacaaagcuuccggcaaacuccgucuugucaggcccacucggcaagucaucgacgucauucacauacacaucaaacuucacagccacgucccugucaaacucgauucccucaaucaccaauaucuccuccucauccuccuuggccuucuugcuccucuucuucugcuucggucucuucaccaccgugcuuaucuucgaccccagacuuaucgggaaacucagugccugccacaacccuugcuggacgacgcacaagcugcgugugcaacucccagcuucuucgcuacuuu 3'。
Claims (2)
1.一种抑制蓝莓多酚氧化酶活性的反义核酸，其特征在于，所述反义核酸为正义链和反义链两条完全互补配对单链组成的双链，所述正义链和所述反义链均由483个碱基组成，所述正义链序列为：5’
aaaguagcgaagaagcugggaguugcacacgcagcuugugcgucguccagcaaggguuguggcaggcacugaguuucccgauaagucuggggucgaagauaagcacgguggugaagagaccgaagcagaagaagaggagcaagaaggccaaggaggaugaggaggagauauuggugauugagggaaucgaguuugacagggacguggcugugaaguuugauguguaugugaaugacgucgaugacuugccgagugggccugacaagacggaguuugccggaagcuuuguaagugugccgcacagccacaagcacaagaagaagaugaacacuauuuugagguuaggguugacagauuuguuggaggaaauugaggcggaggacgaugacagcgugguggugacuuuggugcccaaguucggcgcugucaagauuggugguaucaagauugaauuugcuuuuuaguuaguuaugucaacccagg3’，
所述反义链序列为：5’
ccuggguugacauaacuaacuaaaaagcaaauucaaucuugauaccaccaaucuugacagcgccgaacuugggcaccaaagucaccaccacgcugucaucguccuccgccucaauuuccuccaacaaaucugucaacccuaaccucaaaauaguguucaucuucuucuugugcuuguggcugugcggcacacuuacaaagcuuccggcaaacuccgucuugucaggcccacucggcaagucaucgacgucauucacauacacaucaaacuucacagccacgucccugucaaacucgauucccucaaucaccaauaucuccuccucauccuccuuggccuucuugcuccucuucuucugcuucggucucuucaccaccgugcuuaucuucgaccccagacuuaucgggaaacucagugccugccacaacccuugcuggacgacgcacaagcugcgugugcaacucccagcuucuucgcuacuuu3’。
2.根据权利要求1所述的一种抑制蓝莓多酚氧化酶活性的反义核酸，其在蓝莓组培苗过程中的应用。
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