蜡梅凝集素基因Cplectin在百合中的表达及抗虫性初步鉴定
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摘要
本文以麝香百合(Lilium longiflorum Tbunb)“素雅”(White-Elegance)和“晶体”(Gelria)作为供试材料,通过外植体的再生,抗生素筛选条件及农杆菌介导遗传转化条件的优化,建立了百合的高频再生及遗传转化体系。用农杆菌介导法将蜡梅凝集素基因Cplectin转入百合中,并对转化植株进行了抗虫实验,主要研究结果如下:
1.高频再生体系的建立
(1)“素雅”鳞茎片的诱导分化培养基为MS+6-BA0.5mg/L+NAA0.2mg/L,分化率为91.7%;再生苗鳞茎片的不定芽分化培养基为MS+6-BA1.0mg/L+NAA0.2mg/L,分化率为90%;再生苗叶柄的诱导培养基为MS+NAA1mg/L,分化率为77.5%;生根培养基为1/2MS+NAA0.05 mg/L。
(2)“晶体”鳞茎片的诱导分化培养基为MS+6-BA0.5mg/L+NAA0.5mg/L,分化率为88.3%;愈伤组织的分化培养基为MS+6-BA1.0mg/L+NAA0.1mg/L+蔗糖30g/L+琼脂6g/L,分化率为250%;胚性保持培养基为MS+6-BA0.5mg/L+NAA0.05mg/L+蔗糖30g/L+琼脂6g/L;再生苗鳞茎片的不定芽分化培养基为MS+6-BA 1.0mg/L+NAA0.1mg/L,分化率为87.5%;生根培养基为1/2MS+NAA0.05mg/L。
2.遗传转化体系的建立
(1)受体材料选择
对于“素雅”这一品种再生苗鳞茎片和小叶柄分化率都比较高,但鳞茎片的组织对农杆菌的耐受能力强,且分化能力强,所以得到的抗性芽率高,而转化受体假阳性率也比较高,会增加后期检测的工作量;而再生苗叶柄的再生能力也比较强,且取材方便,多数直接能分化出幼苗,能够降低变异的发生频率,所以选择“素雅”再生苗叶柄作为受体材料。
对于“晶体”这一品种来说,在鳞片再生过程中产生大量的疏松的浅黄色的胚性愈伤组织,多次继代后,仍保持良好的胚性及较强的生命力,接入分化培养基中能顺利分化出再生小植株。这对于遗传转化是非常有利的,所以选择“晶体”胚性愈伤组织作为受体材料。
(2)抗生素浓度的确定
根据卡那霉素(Kan)抗性筛选试验,确定了受体材料适宜的筛选浓度,以“素雅”的再生苗叶柄为受体材料,当卡那霉素的浓度为100mg/L时,叶柄的褐变率为97.5%,为亚致死浓度,所以Kan100mg/L适合作为叶柄的抗性筛选压;同理,以“晶体”的愈伤组织为受体材料时,当卡那霉素浓度为75mg/L时,愈伤组织褐变率为95%,所以Kan75mg/L适合作为愈伤组织的抗性筛选压。在生根培养基中,当Kan浓度为150mg/L时,以上两个品种的再生苗叶全部变黄,没有根生成,所以选用Kan150mg/L作为转化植株生根的筛选浓度。
根据头孢霉素(Cef)抑菌试验,在头孢霉素浓度为250mg/L时,以上受体材料均有很好的抑菌效果,且不影响受体生长,所以选择为250mg/L作为抑菌浓度。
(3)遗传转化条件的研究
本试验对影响农杆菌转化效率的几个主要因素进行了研究,结果发现:取“素雅”的再生苗叶柄作为受体材料,预培养2d后,在OD_(600)值为0.6-0.8的农杆菌菌液的MS(无NH_4NO_3)重悬液中侵染10-12min,再共培养3d,可获得的较高瞬时表达率,为41.6%;“晶体”愈伤组织作为受体时,将经过2-3min创伤处理的愈伤组织,放在OD_(600)值为0.6-0.8的农杆菌菌液的MS重悬液中侵染8-10min。共培养3d,瞬时表达率较高,为43.3%。
在以上研究的基础上,进一步研究发现,两种受体材料,在离心重悬的MS液(无NH_4NO_3)中加入100μmol/L乙酰丁香酮,在共培养基中也加入100μmol/L乙酰丁香酮时,瞬时表达率较不添加乙酰丁香酮时有大幅度提高。分别从41.6%和43.3%,提高到71.6%和75.0%。
3.转化植株的获得
通过筛选培养,获得卡那霉素抗性植株,对“素雅”86株抗性株和“晶体”93株抗性株进行GUS染色和PCR检测,初步鉴定凝集素基因Cplectin已经整合到两种百合中,其中各有21株和25株为阳性。转化率分别为24.4%和26.8%。
4.抗虫性实验
将阳性植株移栽后与未转基因植株的进行抗蚜虫试验,实验结果表明:这两个品种的转基因植株在抗虫性上明显高于对照的未转基因植株。
In this paper,the high-frequency regeneration and the heredity transformation system of Lily were constructed by regenerating the explants,optimizing the antibiotic screening and the Agrobacterium-mediated genetic transformation condition with Lilium longiflorum "White-Elegance" and "Gelria" as tested materials.The Cplectin gene has been successfully recombined into the genomes of lily and the anti-insect experiment was carried on in the transgenic lines.The main results were as following:
1.Establish the high-frequency regeneration system
(1)The results showed that the most suitable medium for inducing the scales of "White-Elegance" to differentiate adventitious buds was MS+6-BA 0.5 mg/L + NAA 0.2 mg/L,the inductivity rate is 91.7%;and the most suitable medium for the scales of the regenerative plants to differentiate adventitious buds was MS+6-BA 1.0 mg/L + NAA 0.2mg/L,the inductivity rate is 90%; the most suitable medium for the petiole of the regenerative plants to differentiate adventitious buds was MS+NAA 1.0 mg/L,the rate is 77.5%.And the rooting medium is 1/2 MS + NAA0.05 mg/L.
(2)The results showed that the most suitable medium for inducing the scales of Lilium longiflorum "Gelria" to differentiate adventitious buds was MS+6-BA 0.5 mg/L+NAA0.05mg/L,the inductivity rate was 88.3%;the best medium for regeneration from callus was MS+6-BA 1.0 mg/L +NAA 0.1 mg/L+ sugar 30g/L+ agar 6g/L,the inductivity rate is 250%;the medium for keeping the embryogenic callus was MS+6-BA 0.5mg/L+NAA 0.05mg/L+ sugar 30g/L+agar 6g/L.And the most suitable medium for the scales of the regenerative plants to differentiate adventitious buds was MS+ 6-BA 1.0 mg/L + NAA 0.1 mg/L,the inductivity rate was 87.5%.And the rooting medium is 1/2 MS +NAA0.05 mg/L.
2.The establishment of genetic transformation system
(1)The selection of receptor material
For "White-Elegance",the inductivity rate of differention for the scales and petiole of the regenreative plants was both high.The scales organization has a strong ability for differention and to resist Agrobacterium,so there would be more resistance buds,but the false positive transformation receptor rate was relatively high.Consequently,it chould increase the workload of later detection. However,the petiole also has strong renewable capacity,and the most of them can regenerate plants.it also can reduce the rate of variation yet,so we choose "White-Elegance" petiole of the regenerative plants as the receptor material.
For "Gelria",there were a lot of loose yellow embryonic calluin in the process of regeneration, and strong vitality could be kept after repeatedly subculture.If put them into the suitable medium for inducing adventitious buds,they could grow out renewable small plants.It was very favorable in genetic transformation,so we choose "Gelria" embryonic callus as the receptor material.
(2)Determining the antibiotics concentration
According to kanamycin(Kan)resistance test,a feasible filtering concentration of receptor materials was determined.Using the "White-Elegance"petiole as the receptor materials,the rate of brown of the petiole was 97.5%with Kan 100 mg/L concentration which was the sublethal concentration,so Kan 100mg/L is suitable as the petiole resistance selection pressure.And also,using "Gelria"callus as the receptor,the rate of brown of the callus was 95.0%with Kan 75mg/L concentration which was the sublethal concentration,so Kan 75mg/L is suitable as the petiole resistance selection pressure.In the rooting culture,when Kan concentration was 150 mg/L,the leaves of the two types of regenerative plants were all yellowing,and there were no roots generation, so we choose Kan 150mg/L as a selection pressure for roots of plants.
According to Cefotamine(Cef)antimicrobial tests,the receptor materials had good antibacterial effects and would not affect the growth of receptor when the concentration of Cef was 250 mg/L, so we choose Cef 250 mg/L as the inhibiting concentration.
(3)Study on conditions of genetic transformation
In the test,several major factors of impacting Wansformation efficiency of Agrobacterium have been studied and results shows that:when using "White-Elegance" petiole as a receptor material, pre-culture 2ds,then put the petiole into the Agrobacterium bacilli with the MS(no NH_4NO_3) re-suspension for 10-12 mins with the OD_(600)of 0.6-0.8 and co-culture 3ds,higher instantaneous rate of 41.6%could be got;when using "Gelria" callus as a receptor,after 2-3 min deal with the trauma, put them into the Agrobacterium bacilli with the MS(no NH_4NO_3)re-suspension for 8-10 mins at the OD_(600)of 0.6-0.8,then co-culture for 3ds,higher instantaneous rate can be reached to 43.3%.
Further study suggested the instantaneous rate of the two receptor is higher when we added 100 μmol/L AS into centrifugal re-suspended in the liquid MS(no NH_4NO_3)and put AS of 100μmol/L in the co-culture medium,and the rate is increased from 41.6%and 43.3%to 71.6%and 75.0% respectively.
3.Obtain of the transgenic plants
21 "White-Elegance"and 25 "Gelria" resistant plants were identified by kanamycin resistance screening,GUS histochemical staining and PCR ananysis to 86 "White-Elegance"and 93 "Gelria". All the analysis suggested that Cplectin gene has been successfully recombined into the genomes of the two material and the conversion rates respectively are 24.4%and 26.8%.
4.Anti-insect experiment
For the anti-aphid tests,the results showed that:the two kinds of transgenic plants has higher insect-resistant than comparision after planting the positive plants
1.高频再生体系的建立
(1)“素雅”鳞茎片的诱导分化培养基为MS+6-BA0.5mg/L+NAA0.2mg/L,分化率为91.7%;再生苗鳞茎片的不定芽分化培养基为MS+6-BA1.0mg/L+NAA0.2mg/L,分化率为90%;再生苗叶柄的诱导培养基为MS+NAA1mg/L,分化率为77.5%;生根培养基为1/2MS+NAA0.05 mg/L。
(2)“晶体”鳞茎片的诱导分化培养基为MS+6-BA0.5mg/L+NAA0.5mg/L,分化率为88.3%;愈伤组织的分化培养基为MS+6-BA1.0mg/L+NAA0.1mg/L+蔗糖30g/L+琼脂6g/L,分化率为250%;胚性保持培养基为MS+6-BA0.5mg/L+NAA0.05mg/L+蔗糖30g/L+琼脂6g/L;再生苗鳞茎片的不定芽分化培养基为MS+6-BA 1.0mg/L+NAA0.1mg/L,分化率为87.5%;生根培养基为1/2MS+NAA0.05mg/L。
2.遗传转化体系的建立
(1)受体材料选择
对于“素雅”这一品种再生苗鳞茎片和小叶柄分化率都比较高,但鳞茎片的组织对农杆菌的耐受能力强,且分化能力强,所以得到的抗性芽率高,而转化受体假阳性率也比较高,会增加后期检测的工作量;而再生苗叶柄的再生能力也比较强,且取材方便,多数直接能分化出幼苗,能够降低变异的发生频率,所以选择“素雅”再生苗叶柄作为受体材料。
对于“晶体”这一品种来说,在鳞片再生过程中产生大量的疏松的浅黄色的胚性愈伤组织,多次继代后,仍保持良好的胚性及较强的生命力,接入分化培养基中能顺利分化出再生小植株。这对于遗传转化是非常有利的,所以选择“晶体”胚性愈伤组织作为受体材料。
(2)抗生素浓度的确定
根据卡那霉素(Kan)抗性筛选试验,确定了受体材料适宜的筛选浓度,以“素雅”的再生苗叶柄为受体材料,当卡那霉素的浓度为100mg/L时,叶柄的褐变率为97.5%,为亚致死浓度,所以Kan100mg/L适合作为叶柄的抗性筛选压;同理,以“晶体”的愈伤组织为受体材料时,当卡那霉素浓度为75mg/L时,愈伤组织褐变率为95%,所以Kan75mg/L适合作为愈伤组织的抗性筛选压。在生根培养基中,当Kan浓度为150mg/L时,以上两个品种的再生苗叶全部变黄,没有根生成,所以选用Kan150mg/L作为转化植株生根的筛选浓度。
根据头孢霉素(Cef)抑菌试验,在头孢霉素浓度为250mg/L时,以上受体材料均有很好的抑菌效果,且不影响受体生长,所以选择为250mg/L作为抑菌浓度。
(3)遗传转化条件的研究
本试验对影响农杆菌转化效率的几个主要因素进行了研究,结果发现:取“素雅”的再生苗叶柄作为受体材料,预培养2d后,在OD_(600)值为0.6-0.8的农杆菌菌液的MS(无NH_4NO_3)重悬液中侵染10-12min,再共培养3d,可获得的较高瞬时表达率,为41.6%;“晶体”愈伤组织作为受体时,将经过2-3min创伤处理的愈伤组织,放在OD_(600)值为0.6-0.8的农杆菌菌液的MS重悬液中侵染8-10min。共培养3d,瞬时表达率较高,为43.3%。
在以上研究的基础上,进一步研究发现,两种受体材料,在离心重悬的MS液(无NH_4NO_3)中加入100μmol/L乙酰丁香酮,在共培养基中也加入100μmol/L乙酰丁香酮时,瞬时表达率较不添加乙酰丁香酮时有大幅度提高。分别从41.6%和43.3%,提高到71.6%和75.0%。
3.转化植株的获得
通过筛选培养,获得卡那霉素抗性植株,对“素雅”86株抗性株和“晶体”93株抗性株进行GUS染色和PCR检测,初步鉴定凝集素基因Cplectin已经整合到两种百合中,其中各有21株和25株为阳性。转化率分别为24.4%和26.8%。
4.抗虫性实验
将阳性植株移栽后与未转基因植株的进行抗蚜虫试验,实验结果表明:这两个品种的转基因植株在抗虫性上明显高于对照的未转基因植株。
In this paper,the high-frequency regeneration and the heredity transformation system of Lily were constructed by regenerating the explants,optimizing the antibiotic screening and the Agrobacterium-mediated genetic transformation condition with Lilium longiflorum "White-Elegance" and "Gelria" as tested materials.The Cplectin gene has been successfully recombined into the genomes of lily and the anti-insect experiment was carried on in the transgenic lines.The main results were as following:
1.Establish the high-frequency regeneration system
(1)The results showed that the most suitable medium for inducing the scales of "White-Elegance" to differentiate adventitious buds was MS+6-BA 0.5 mg/L + NAA 0.2 mg/L,the inductivity rate is 91.7%;and the most suitable medium for the scales of the regenerative plants to differentiate adventitious buds was MS+6-BA 1.0 mg/L + NAA 0.2mg/L,the inductivity rate is 90%; the most suitable medium for the petiole of the regenerative plants to differentiate adventitious buds was MS+NAA 1.0 mg/L,the rate is 77.5%.And the rooting medium is 1/2 MS + NAA0.05 mg/L.
(2)The results showed that the most suitable medium for inducing the scales of Lilium longiflorum "Gelria" to differentiate adventitious buds was MS+6-BA 0.5 mg/L+NAA0.05mg/L,the inductivity rate was 88.3%;the best medium for regeneration from callus was MS+6-BA 1.0 mg/L +NAA 0.1 mg/L+ sugar 30g/L+ agar 6g/L,the inductivity rate is 250%;the medium for keeping the embryogenic callus was MS+6-BA 0.5mg/L+NAA 0.05mg/L+ sugar 30g/L+agar 6g/L.And the most suitable medium for the scales of the regenerative plants to differentiate adventitious buds was MS+ 6-BA 1.0 mg/L + NAA 0.1 mg/L,the inductivity rate was 87.5%.And the rooting medium is 1/2 MS +NAA0.05 mg/L.
2.The establishment of genetic transformation system
(1)The selection of receptor material
For "White-Elegance",the inductivity rate of differention for the scales and petiole of the regenreative plants was both high.The scales organization has a strong ability for differention and to resist Agrobacterium,so there would be more resistance buds,but the false positive transformation receptor rate was relatively high.Consequently,it chould increase the workload of later detection. However,the petiole also has strong renewable capacity,and the most of them can regenerate plants.it also can reduce the rate of variation yet,so we choose "White-Elegance" petiole of the regenerative plants as the receptor material.
For "Gelria",there were a lot of loose yellow embryonic calluin in the process of regeneration, and strong vitality could be kept after repeatedly subculture.If put them into the suitable medium for inducing adventitious buds,they could grow out renewable small plants.It was very favorable in genetic transformation,so we choose "Gelria" embryonic callus as the receptor material.
(2)Determining the antibiotics concentration
According to kanamycin(Kan)resistance test,a feasible filtering concentration of receptor materials was determined.Using the "White-Elegance"petiole as the receptor materials,the rate of brown of the petiole was 97.5%with Kan 100 mg/L concentration which was the sublethal concentration,so Kan 100mg/L is suitable as the petiole resistance selection pressure.And also,using "Gelria"callus as the receptor,the rate of brown of the callus was 95.0%with Kan 75mg/L concentration which was the sublethal concentration,so Kan 75mg/L is suitable as the petiole resistance selection pressure.In the rooting culture,when Kan concentration was 150 mg/L,the leaves of the two types of regenerative plants were all yellowing,and there were no roots generation, so we choose Kan 150mg/L as a selection pressure for roots of plants.
According to Cefotamine(Cef)antimicrobial tests,the receptor materials had good antibacterial effects and would not affect the growth of receptor when the concentration of Cef was 250 mg/L, so we choose Cef 250 mg/L as the inhibiting concentration.
(3)Study on conditions of genetic transformation
In the test,several major factors of impacting Wansformation efficiency of Agrobacterium have been studied and results shows that:when using "White-Elegance" petiole as a receptor material, pre-culture 2ds,then put the petiole into the Agrobacterium bacilli with the MS(no NH_4NO_3) re-suspension for 10-12 mins with the OD_(600)of 0.6-0.8 and co-culture 3ds,higher instantaneous rate of 41.6%could be got;when using "Gelria" callus as a receptor,after 2-3 min deal with the trauma, put them into the Agrobacterium bacilli with the MS(no NH_4NO_3)re-suspension for 8-10 mins at the OD_(600)of 0.6-0.8,then co-culture for 3ds,higher instantaneous rate can be reached to 43.3%.
Further study suggested the instantaneous rate of the two receptor is higher when we added 100 μmol/L AS into centrifugal re-suspended in the liquid MS(no NH_4NO_3)and put AS of 100μmol/L in the co-culture medium,and the rate is increased from 41.6%and 43.3%to 71.6%and 75.0% respectively.
3.Obtain of the transgenic plants
21 "White-Elegance"and 25 "Gelria" resistant plants were identified by kanamycin resistance screening,GUS histochemical staining and PCR ananysis to 86 "White-Elegance"and 93 "Gelria". All the analysis suggested that Cplectin gene has been successfully recombined into the genomes of the two material and the conversion rates respectively are 24.4%and 26.8%.
4.Anti-insect experiment
For the anti-aphid tests,the results showed that:the two kinds of transgenic plants has higher insect-resistant than comparision after planting the positive plants
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