甜叶菊品系“1096”离体培养及其UGT76G1酶基因的克隆
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摘要
我国甜叶菊品种是80年代初开始引种的,由于该植物自交不育异花授粉,几代杂合之后植株良莠不齐,需要筛选新的优良品种,同时需要改进种植方式。采用组织培养技术不仅能保持品种的优良特性,还可不受外界条件的影响周年繁殖,在较短时间内即可繁育出大批种苗;利用组织培养技术还将推动甜叶菊的遗传改良,弥补甜叶菊传统育种的不足。世界卫生组织和联合国粮食组织(WHO/FAO)所属的食品添加剂专家委员会(JECFA)在第63次日内瓦会议上认可了甜叶菊提取物使用的试行案。即人的体重每公斤每天可摄取0～2mg甜叶菊糖苷,同时规定糖苷的纯度应该大于95%,这就要求我们改进生产工艺降低耗水量,增加纯度,特别是生产口味纯正的RebaudiosideA(RA)含量高的产品,或培育出高RA含量的甜叶菊新品种。
本试验在前人研究的基础上,建立了本实验室采用改良DNS等方法筛选出的,高糖苷含量品系“1096”的组培快繁体系,为该品系在生产上的推广应用奠定基础;并探讨了不同浓度蔗糖和甘露醇对其离体保存的影响,为控制增殖继代次数及甜叶菊优良种质的离体保存提供理论基础:采用TD-PCR法克隆了控制由Steviolbioside和Stevioside到RebaudiosideA合成的UGT76G1酶的基因。主要研究结果如下:
1.采用甜叶菊品系“1096”为材料,研究了不同外植体、不同激素种类和配比对愈伤组织诱导、不定芽分化,生根及移栽的影响。结果表明:以茎尖为外植体愈伤组织和不定芽诱导率均最高;其次是带腋芽茎段;再次为叶片。无腋芽茎段和无菌根均能诱导出愈伤组织,但不能诱导成芽。茎尖不定芽诱导的最佳培养基为MS+6-BA 1.0mg/L;叶片不定芽诱导的最佳培养基为MS+6-BA 1.0mg/L+IAA 1.0mg/L;带腋芽茎段不定芽诱导的最佳培养基为MS+6-BA1.5mg/L。不定芽继代增殖培养采用MS+6-BA 0.5mg/L+NAA 0.05mg/L;在1/4MS+IBA0.1mg/L+NAA 0.1mg/L+1g/L活性炭的培养基上诱导生根,生根率可达100%,生根苗移栽后成活率达95%。
2.甜叶菊种质离体培养保存具有一定的可行性。1/4MS+10g/L甘露醇更适合于甜叶菊品系“1096”的离体保存,试管苗经过再生培养,均能恢复原有的形态,其叶型、株型等形态特征与对照没有差别。
3.利用反转录与TD-PCR技术克隆了甜叶菊品系“1096”UGT76G1酶的基因,并利用部分生物信息学软件对克隆出的基因和推导的氨基酸序列进行了分析,为今后利用生物工程手段改善甜叶菊糖苷味质及培育高RA含量的甜叶菊新品种等奠定基础。
Stevia rebaudiana is introduced into China Since 1980's,for Stevia rebaudiana is a cross-pollination plant,steviol glycosides content in its leaves varies greatly after several years,key to up question is to reform growing method and to select new culturer,to get more and better leaves, we try to develop new method to select better strains.The tissue culture technology not only to be able to maintain the good characteristics of Stevia rebaudiana,but may also propagate quickly and breed large quantities of seedlings,In a short time,which are not required to consider environic condition.Using tissue culture technology will also promote genetic improvement and make up for the deficiency of traditional breeding method of Stevia rebaudiana.
Steviol glycosides,which material containing not less than 95%,are approved to be used as addictive at the sixty-third meeting of the Joint FAO/WHO Expert Committee on Food Additives (JECFA).The Committee established a temporary ADI of 0～2 mg/kg bw for steviol glycosides,so key to the question of stevia industry is to reform extract method,to improve Purity,to decrease production costs,to product high RebaudiosideA content steviol glycosides with good taste,or to culture stevia rebaudiana new variety with high RA content characteristic.
In the present study,we established rapid propagation system of stevia rebaudiana strain "1096" which screened by improved DNS method in our laboratory.We discussed the influence of the different density sucrose and mannitol on "1096" strain in vitro conservation,colned the gene of UGT76G1 which Controls steviolbioside and stevioside to RebaudiosideA..The main experimental results were as follows:
1.The effects of different combinations of growth substances and explants on callus induction, bud differentiation,root formation and transplant were investigated in Stevia rebaudiana strain "1096".The results showed that stem apex seemed to be the best explant for callus induction and bud differentiation,followed by stem segment with axillary bud and the third was leaf.Both the stem segment without axillary bud and root of plantlets could be induced to form calli but not adventitious buds.The optimal combinations of 6-BA for adventitious bud induction on stem apex was 1.0mg/L,on stem segment with axillary bud was 1.5mg/L on MS medium;The best adventitious buds induction medium from leaves was MS medium with 1.0mg/L 6-BA and 1.0mg/L IAA.The proliferation culture medium for bud differentiation was MS medium with 0.5 mg/L 6-BA and 0.05 mg/L NAA.Rooting medium is 1/4MS+IBA 0.1 mg/L+NAA 0.1mg/L +active carbon 1.0g/L,the rooting rate reached up to 100%,transplant survive was 95%.
2.1t is feasible to conserve the stevia rebaudiana germplasm in vitro.The 1/4MS medium with 10g/L mannitol is better for "1096"strain vitro conservation than sucrose.After regeneration culture, the plantlet can restore the original shape,there is no difference in leaf shape and plant type between the plantlet of recovering growth and control group.
3.Clone gene of UGT76G1 of "1096"strain by reverse transcription and touchdown PCR. Cloning product and its encoded protein was analyzed with bioinformatics software.Lay a foundation for improving taste of steviol glycosides,the further breeding of high RA content new stevia rebaudiana Variety by genetic engineering method.
本试验在前人研究的基础上,建立了本实验室采用改良DNS等方法筛选出的,高糖苷含量品系“1096”的组培快繁体系,为该品系在生产上的推广应用奠定基础;并探讨了不同浓度蔗糖和甘露醇对其离体保存的影响,为控制增殖继代次数及甜叶菊优良种质的离体保存提供理论基础:采用TD-PCR法克隆了控制由Steviolbioside和Stevioside到RebaudiosideA合成的UGT76G1酶的基因。主要研究结果如下:
1.采用甜叶菊品系“1096”为材料,研究了不同外植体、不同激素种类和配比对愈伤组织诱导、不定芽分化,生根及移栽的影响。结果表明:以茎尖为外植体愈伤组织和不定芽诱导率均最高;其次是带腋芽茎段;再次为叶片。无腋芽茎段和无菌根均能诱导出愈伤组织,但不能诱导成芽。茎尖不定芽诱导的最佳培养基为MS+6-BA 1.0mg/L;叶片不定芽诱导的最佳培养基为MS+6-BA 1.0mg/L+IAA 1.0mg/L;带腋芽茎段不定芽诱导的最佳培养基为MS+6-BA1.5mg/L。不定芽继代增殖培养采用MS+6-BA 0.5mg/L+NAA 0.05mg/L;在1/4MS+IBA0.1mg/L+NAA 0.1mg/L+1g/L活性炭的培养基上诱导生根,生根率可达100%,生根苗移栽后成活率达95%。
2.甜叶菊种质离体培养保存具有一定的可行性。1/4MS+10g/L甘露醇更适合于甜叶菊品系“1096”的离体保存,试管苗经过再生培养,均能恢复原有的形态,其叶型、株型等形态特征与对照没有差别。
3.利用反转录与TD-PCR技术克隆了甜叶菊品系“1096”UGT76G1酶的基因,并利用部分生物信息学软件对克隆出的基因和推导的氨基酸序列进行了分析,为今后利用生物工程手段改善甜叶菊糖苷味质及培育高RA含量的甜叶菊新品种等奠定基础。
Stevia rebaudiana is introduced into China Since 1980's,for Stevia rebaudiana is a cross-pollination plant,steviol glycosides content in its leaves varies greatly after several years,key to up question is to reform growing method and to select new culturer,to get more and better leaves, we try to develop new method to select better strains.The tissue culture technology not only to be able to maintain the good characteristics of Stevia rebaudiana,but may also propagate quickly and breed large quantities of seedlings,In a short time,which are not required to consider environic condition.Using tissue culture technology will also promote genetic improvement and make up for the deficiency of traditional breeding method of Stevia rebaudiana.
Steviol glycosides,which material containing not less than 95%,are approved to be used as addictive at the sixty-third meeting of the Joint FAO/WHO Expert Committee on Food Additives (JECFA).The Committee established a temporary ADI of 0～2 mg/kg bw for steviol glycosides,so key to the question of stevia industry is to reform extract method,to improve Purity,to decrease production costs,to product high RebaudiosideA content steviol glycosides with good taste,or to culture stevia rebaudiana new variety with high RA content characteristic.
In the present study,we established rapid propagation system of stevia rebaudiana strain "1096" which screened by improved DNS method in our laboratory.We discussed the influence of the different density sucrose and mannitol on "1096" strain in vitro conservation,colned the gene of UGT76G1 which Controls steviolbioside and stevioside to RebaudiosideA..The main experimental results were as follows:
1.The effects of different combinations of growth substances and explants on callus induction, bud differentiation,root formation and transplant were investigated in Stevia rebaudiana strain "1096".The results showed that stem apex seemed to be the best explant for callus induction and bud differentiation,followed by stem segment with axillary bud and the third was leaf.Both the stem segment without axillary bud and root of plantlets could be induced to form calli but not adventitious buds.The optimal combinations of 6-BA for adventitious bud induction on stem apex was 1.0mg/L,on stem segment with axillary bud was 1.5mg/L on MS medium;The best adventitious buds induction medium from leaves was MS medium with 1.0mg/L 6-BA and 1.0mg/L IAA.The proliferation culture medium for bud differentiation was MS medium with 0.5 mg/L 6-BA and 0.05 mg/L NAA.Rooting medium is 1/4MS+IBA 0.1 mg/L+NAA 0.1mg/L +active carbon 1.0g/L,the rooting rate reached up to 100%,transplant survive was 95%.
2.1t is feasible to conserve the stevia rebaudiana germplasm in vitro.The 1/4MS medium with 10g/L mannitol is better for "1096"strain vitro conservation than sucrose.After regeneration culture, the plantlet can restore the original shape,there is no difference in leaf shape and plant type between the plantlet of recovering growth and control group.
3.Clone gene of UGT76G1 of "1096"strain by reverse transcription and touchdown PCR. Cloning product and its encoded protein was analyzed with bioinformatics software.Lay a foundation for improving taste of steviol glycosides,the further breeding of high RA content new stevia rebaudiana Variety by genetic engineering method.
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