甘蓝型油菜KCS基因家族表达及功能分析
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摘要
KCS基因家族编码β-酮脂酰CoA合成酶，催化超长链脂肪酸延伸过程的缩合反应，是超长链脂肪酸合成的限速酶。通过克隆KCS基因，可阐明KCS基因在超长链脂肪酸及蜡质合成中的功能，为油菜脂肪酸改良提供新的基因源。
本研究采用同源序列法分离了高芥酸油菜品种中油821、零芥酸野生种诸葛菜和荠菜，含芥酸野生种野芥、白芥和菘蓝中的FAE1基因序列。5个野生种的FAE1基因序列与甘蓝型油菜中油821的FAE1基因同源性高于85%。在酵母中异源表达野生种的FAE1基因，各FAE1基因都可正常表达，转化诸葛菜和荠菜FAE1基因的酵母不能合成超长链脂肪酸，而转化野芥、白芥和菘蓝的FAE1基因的酵母都有微量长链脂肪酸的合成。实验结果表明诸葛菜和芥菜的低芥酸性状源于FAE1基因编码产物的失活。无论是高芥酸物种还是零芥酸物种，其FAE1基因的核苷酸序列高度保守。
FAE1基因是芥酸合成的限速酶，通过定向抑制FAE1基因的功能，有望将高芥酸油菜品种改良为低芥酸油菜品种。选用FAE1基因的全长序列构建FAE1基因的RNAi载体。将FAE1基因编码区以头对头的方式构建到种子特异性Napin启动子的下游，反向重复的FAE1序列用组蛋白H2A内含子间隔。采用农杆菌介导法将构建的FAE1RNAi载体转化到高芥酸甘蓝型油菜品种中油821中，获得81株转化苗，其中有51株阳性苗。用气相色谱法分析转化株T1代混合种子的脂肪酸组成，受体品种中油821的芥酸含量为43.35%，转基因植株的芥酸含量与野生型相比有不同程度的降低，21株芥酸含量低于35%，2个转化株芥酸含量在10-20%之间。用近红外光谱仪分析转基因植株T2代种子，多数株系的芥酸含量与高芥酸油菜无明显差异，只有1个株系的后代其芥酸性状符合1:3的分离比，低芥酸植株的芥酸含量最低达到2%的水平。结果表明，通过RNAi技术介导FAE1基因沉默可以有效的降低转基因植株中的芥酸含量，但不能降至零芥酸水平。低芥酸转基因植株具有明显的非预期效应，表现出种子皱缩、形状不规则、种皮没有光泽而且难以正常生长等。
在前期基因组步移及RACE实验的基础上，克隆了BnKCS1、BnKCS3、BnKCS4、BnKCS5、BnKCS6、BnKCS8、BnKCS9、BnKCS10、BnKCS11、BnKCS12、BnKCS13、BnKCS15、BnKCS16、BnKCS17、BnKCS19、BnKCS20和BnKCS21共17个基因的编码序列。对分离的BnKCS基因成员进行亲缘关系分析，从甘蓝型油菜中分离的BnKCS基因成员可分为6个亚类。FAE1基因与KCS基因家族其他成员间的序列同源性在45.6%-63.1%之间，BnKCS4、BnKCS8、BnKCS9、BnKCS16和BnKCS17基因与FAE1基因属于同一亚类，它们与FAE1基因的序列一致性均大于60%。BnKCS基因的表达谱分析表明，种子中表达的基因有9个，包括BnKCS1、BnKCS4、BnKCS5、BnKCS6、BnKCS9、BnKCS10、BnKCS11、BnKCS19和BnKCS20，这些基因与FAE1基因的同源性均大于50%。推测用FAE1基因的全长序列构建FAE1RNAi载体，不仅抑制FAE1基因的表达，还会协同抑制其他同源基因的表达，从而产生非预期效应。需在充分的分析KCS基因家族的基因序列的基础上，利用FAE1基因的特异性序列构建RNAi载体或MicroRNAi载体，达到特异性抑制FAE1基因表达的目的。
用KCS基因家族的保守结构域FAE1-CUT1-RPPA检索甘蓝型油菜的全基因组序列，检索到了40条KCS基因序列。但未在甘蓝型油菜中检索KCS3、KCS7、KCS8、KCS11、KCS12、KCS16和KCS21基因的同源序列。而在本研究中，我们通过同源序列法从甘蓝型油菜中分离到了BnKCS3、BnKCS8、BnKCS11、BnKCS12、BnKCS16和BnKCS21基因。推测，甘蓝型油菜中KCS基因家族可能至少有46个成员。
用实时荧光的方法分析甘蓝型油菜中BnKCS基因在根、茎、叶、花蕾、柱头、花粉、30d荚果皮、40d荚果皮、30d种子和40d种子中的表达量。各BnKCS基因在甘蓝型油菜不同组织或器官中具有不同的表达模式，暗示各个基因在不同的组织或器官行使功能。所分析的18个BnKCS基因都在油菜花器官中表达，10个BnKCS基因在茎中表达，13个基因在叶中表达，9个基因在种子中有表达，8个基因在角果皮中表达。BnKCS2、BnKCS7、BnKCS8、BnKCS9和BnKCS215个基因在花器官中特异表达，其中BnKCS2和BnKCS9只在花粉中特异性表达，BnKCS21仅在花瓣中特异表达。
将克隆的17个BnKCS基因构建到酵母表达载体pYES2/NTA中半乳糖诱导启动子的下游，在酵母中进行异源表达分析。15个BnKCS基因在酵母中正常表达并翻译蛋白，BnKCS3和BnKCS4受密码子偏好的影响在酵母中没有表达产物。分析酵母脂肪酸组成发现，只有转BnKCS1基因和FAE1基因的酵母中有超长链脂肪酸的合成，其它转基因酵母均没有新的超长链脂肪酸合成。结果表明这些BnKCS基因不能利用酵母中的脂肪酸作为催化底物。在后续研究中需要在酵母体外添加超长链脂肪酸做底物，进一步研究BnKCS基因的功能。
为了揭示KCS基因与蜡质合成的相关性，鉴定出了7个有突变体纯系的拟南芥kcs突变体，包括kcs4、kcs7、kcs9、kcs11、kcs13、kcs16和kcs17。基因表达谱分析显示，突变体中的靶标基因因T-DNA的插入而沉默。用扫描电镜扫描拟南芥突变体及野生型角果和茎表面的蜡质晶体密度，突变体与野生型相比，茎和角果表面的蜡质晶体密度没有明显变化。通过GC-MS分析kcs突变体角果、茎和叶的表面蜡质组成。发现除kcs4突变体外，其余的kcs突变体茎、角果和叶的部分蜡质组分与野生型相比有显著降低，且链长不一，既有酰基还原途径的产物，如初级醇，也有脱羰基途径的产物如烷烃、醛和29酮。kcs突变体没有出现出蜡质严重缺失的性状，也没出现某一链长的超长链脂肪酸衍生物过度积累或某一蜡质组分完全缺失的情况。表明本研究中的KCS7、KCS8、KCS9、KCS11、KCS13、KCS16和KCS17基因的催化功能与其他KCS基因相互重叠，催化产物不是某一特定链长的超长链脂肪酸，而且催化产物既参与酰基还原途径也参与脱羰基途径的代谢。
KCS gene family encode β-ketoacyl CoA synthase, a rate-limiting enzyme in the synthesis of very long chain fatty acid (VLCFA), catalyzing the condensation reaction of VLCFA elongation process. Cloning of KCSgenes help to elucidate the function of the KCSgenes in the synthesis of VLCFAs and wax, and provide a new source of genes for improving rapeseed fatty acid composition.
In this study, FAE1gene sequences were isolated from high erucic acid B. napus varieties Zhongyou821, zero-erucic acid wild species Orychophragmus violaceus L.and Capsella bursapastrois L., wild species containing erucic acid Sinapis alba L., Sinapis arvensis L. and Isatis indigotica Fort. Comparison of FAE1sequences indicated that the FAE1gene of Zhongyou821showed more than85%identity with that of five wild species. Heterologous expression of the FAE1genes was performed in the yeast cell, all the FAE1genes from the wild cruciferous species can normally express in yeast cells. The yeast cells expressing FAE1genes from O. violaceus and C. buraspastroil showed no VLCFA formation, and those expressing FAE1genes from S. arvensis, S. alba, and I. indigotica produced trace amount VLCFA. Experimental results show that the low erucic acid trait of O. violaceus and C. buraspastroil comes from the inactivation of protein activity encoded by FAE1gene. The FAE1nucleotide sequences are highly conserved between high erucic acid species and zero erucic acid species.
FAE1gene is the rate-limiting enzyme in the synthesis of erucic acid. The high erucic acid rapeseed varieties can be improved for low erucic acid rapeseed varieties through directed inhibition of FAE1gene function. The full-length sequence of FAE1gene was chosen to construct the FAE1RNAi vector. The FAE1sequences were cloned to downstream of the seed-specific Napin promoter as a head-to-head concatemer, and the inverted repeats of FAE1sequence were spaced apart by a histone H2A intron. The FAE1RNAi vector was transformed into high erucic acid B. napus varieties Zhongyou821by Agrobacterium-mediated method. After transformation,81transformants were obtained, in which,51positive seedlings were identified by PCR method. GC analysis of fatty acid composition of transgenic T1generation seeds revealed that the erucic acid content of transgenic rapeseed decreased in different degree compared with the wild type. The erucic acid content of recipient varieties Zhongyou821was43.35%, that of21transformants is less than35%, in which, the erucic acid content of two transformants decrease to10-20%. Near-infrared analysis of T2generation seeds showed that the erucic acid content of most lines displayed no significant difference compared with high erucic acid rapeseed. The erucic acid trait displayed separation ratio of1:3for the descendants of one line, the erucic acid content of low erucic acid transgenic plant was at the level of2%. The results revealed that the FAE1silence mediated by RNAi technology can effectively reduce the erucic acid content of transgenic plants, but can not reduce to zero erucic acid level. And low erucic acid transformants have obvious unintended effects, showing seed shrinkage, irregular shape, seed coat glossless and difficult to normally grow.
On the basis of previous genomewalking and RACE experiment, we cloned17KCS gene members, including BnKCS1, BnKCS3, BnKCS4, BnKCS5, BnKCS6, BnKCS8, BnKCS9, BnKCS10, BnKCS11, BnKCS12, BnKCS13, BnKCS15, BnKCS16, BnKCS17, BnKCSl9, BnKCS20and BnKCS21. According to the pedigree analysis, BnKCS gene members can be divided into six sub-categories. The sequence homology ranges from45.6%to63.1%between the FAE1gene and other members of the KCS gene family. BnKCS4, BnKCS8, BnKCS9, BnKCS16, BnKCS17and FAE1genes belong to one sub-category, these genes show more than60%identity with FAE1gene. Expression profile analysis of BnKCS genes showed that there are9BnKCS genes expressing in the seeds, including BnKCS1, BnKCS4, BnKCSS, BnKCS6, BnKCS9, BnKCS10, BnKCS11, BnKCS19and BnKCS20. these genes exhibit greater than50%homology with FAE1gene. It was speculated that the FAE1full-length sequence was used to construct FAE1RNAi vector, which not only inhibit the expression of FAE1gene, but also synergisticlly inhibit the expression of homologous genes, thus, resulting in unintended effects. In the future, it is better to identify FAE1specific sequence for constructing FAE1RNAi or MicroRNAi vector on the basis of a comprehensive analysis of the sequences of the KCS gene family, to achieve the specific purpose of inhibiting the function of the FAE1gene.
The conserved domain FAEl-CUT1-RPPA of KCS family was usend to search the KCS sequences across the whole genomic sequence of B. napus, and40KCS sequences were obtained. Wherereas, no homologous sequence of KCS3, KCS7, KCS8, KCS11, KCS12, KCS16and KCS21were retrieved. In this study, BnKCS3, BnKCS8, BnKCS11, Bn KCS12, BnKCS16and BnKCS21were isolated form B. napus. It is concluded that the KCS family contains46gene members at least in B, napus.
The real-time PCR method was used to analyze the expression profile of BnKCS genes in different tissues of B. napus, including root, stem, leaf, bud, stigma, pollen,30d pod skin,40d pod skin,30d seeds and40d seed. The BnKCS genes have different expression pattern in different tissues or organs. The analysis results shows that18BnKCS genes are expressed in the floral organ of B. napus,10BnKCS genes expressed in the stem,13BnKCS genes expressed in leaves,9genes expressed in seed,8genes expressed in the pod skin.5genes (BnKCS2, BnKCS7, BnKCS8, BnKCS9and BnKCS21) are specifically expressed in floral organ, of which, BnKCS2and BnKCS9are specially expressed in the pollen, BnKCS21is specifically expressed in the petals.
17BnKCS genes were cloned to downstream of the galactose-inducible promoter of the yeast expression vector pYES2/NT, to perform heterologous expression in yeast cells.15BnKCS gene can normally translate protein, and the BnKCS3and the BnKCS4fail to be expressed due to the codon preference. GC analysis of yeast fatty acid composition indicated that the VLCFAs were only observed in yeast expressing the BnKCS1and FAE1genes, and no VLCFA synthesis in yeast expressing other BnKCS genes. It was concluded that these BnKCS genes can not take advantage of the fatty acids in yeast as a catalytic substrate to synthesize VLCFA. In follow-up studies, VLCFAs should be added to the yeast medium in vitro for further studying of BnKCS gene function.
To reveal the relationship between the KCS genes and wax biosynthesis, the homozygotes of7Arabidopsis Kcs mutant were identified, including kcs4, kcs7kcs9, kcsll kcs13, kcs16and kcs17. The KCS gene expression profile analysis showed that the target gene in the mutant was silenced due to the insertion of the T-DNA. The wax crystal density of the surface of Arabidopsis siliques and stems were scanned using a scanning electron microscope, and found that the wax crystal density of the mutant does not significantly change compared with that of the wild type. GC-MS was carried out to analyze the wax composition of Arabidopsis siliques, stems and leaves. Compared to the wild-type, part of the wax components were significantly reduced in stems, siliques and leaves for kcs mutants with except of the kcs4mutant. And the wax components with significant change have different chain length, including both the product of the acyl reduction pathway, such as primary alcohols, and that of decarbonylation pathway, such as alkanes, aldehydes and29C ketone. The kcs mutant did not appear serious deficiencies of surface wax, or excessive accumulation of a derivative of VLCFA and total loss of someone wax composition. This study revealed that the catalytic function was overlapping with other KCS gene for the KCS7, KCS8, KCS9, ACS11, KCS13, KCS16and KCS17genes, their catalytic product is not a VLCFA with specific chain length, involved in the metabolism of both acyl reduction pathway and decarbonylation pathway.
本研究采用同源序列法分离了高芥酸油菜品种中油821、零芥酸野生种诸葛菜和荠菜，含芥酸野生种野芥、白芥和菘蓝中的FAE1基因序列。5个野生种的FAE1基因序列与甘蓝型油菜中油821的FAE1基因同源性高于85%。在酵母中异源表达野生种的FAE1基因，各FAE1基因都可正常表达，转化诸葛菜和荠菜FAE1基因的酵母不能合成超长链脂肪酸，而转化野芥、白芥和菘蓝的FAE1基因的酵母都有微量长链脂肪酸的合成。实验结果表明诸葛菜和芥菜的低芥酸性状源于FAE1基因编码产物的失活。无论是高芥酸物种还是零芥酸物种，其FAE1基因的核苷酸序列高度保守。
FAE1基因是芥酸合成的限速酶，通过定向抑制FAE1基因的功能，有望将高芥酸油菜品种改良为低芥酸油菜品种。选用FAE1基因的全长序列构建FAE1基因的RNAi载体。将FAE1基因编码区以头对头的方式构建到种子特异性Napin启动子的下游，反向重复的FAE1序列用组蛋白H2A内含子间隔。采用农杆菌介导法将构建的FAE1RNAi载体转化到高芥酸甘蓝型油菜品种中油821中，获得81株转化苗，其中有51株阳性苗。用气相色谱法分析转化株T1代混合种子的脂肪酸组成，受体品种中油821的芥酸含量为43.35%，转基因植株的芥酸含量与野生型相比有不同程度的降低，21株芥酸含量低于35%，2个转化株芥酸含量在10-20%之间。用近红外光谱仪分析转基因植株T2代种子，多数株系的芥酸含量与高芥酸油菜无明显差异，只有1个株系的后代其芥酸性状符合1:3的分离比，低芥酸植株的芥酸含量最低达到2%的水平。结果表明，通过RNAi技术介导FAE1基因沉默可以有效的降低转基因植株中的芥酸含量，但不能降至零芥酸水平。低芥酸转基因植株具有明显的非预期效应，表现出种子皱缩、形状不规则、种皮没有光泽而且难以正常生长等。
在前期基因组步移及RACE实验的基础上，克隆了BnKCS1、BnKCS3、BnKCS4、BnKCS5、BnKCS6、BnKCS8、BnKCS9、BnKCS10、BnKCS11、BnKCS12、BnKCS13、BnKCS15、BnKCS16、BnKCS17、BnKCS19、BnKCS20和BnKCS21共17个基因的编码序列。对分离的BnKCS基因成员进行亲缘关系分析，从甘蓝型油菜中分离的BnKCS基因成员可分为6个亚类。FAE1基因与KCS基因家族其他成员间的序列同源性在45.6%-63.1%之间，BnKCS4、BnKCS8、BnKCS9、BnKCS16和BnKCS17基因与FAE1基因属于同一亚类，它们与FAE1基因的序列一致性均大于60%。BnKCS基因的表达谱分析表明，种子中表达的基因有9个，包括BnKCS1、BnKCS4、BnKCS5、BnKCS6、BnKCS9、BnKCS10、BnKCS11、BnKCS19和BnKCS20，这些基因与FAE1基因的同源性均大于50%。推测用FAE1基因的全长序列构建FAE1RNAi载体，不仅抑制FAE1基因的表达，还会协同抑制其他同源基因的表达，从而产生非预期效应。需在充分的分析KCS基因家族的基因序列的基础上，利用FAE1基因的特异性序列构建RNAi载体或MicroRNAi载体，达到特异性抑制FAE1基因表达的目的。
用KCS基因家族的保守结构域FAE1-CUT1-RPPA检索甘蓝型油菜的全基因组序列，检索到了40条KCS基因序列。但未在甘蓝型油菜中检索KCS3、KCS7、KCS8、KCS11、KCS12、KCS16和KCS21基因的同源序列。而在本研究中，我们通过同源序列法从甘蓝型油菜中分离到了BnKCS3、BnKCS8、BnKCS11、BnKCS12、BnKCS16和BnKCS21基因。推测，甘蓝型油菜中KCS基因家族可能至少有46个成员。
用实时荧光的方法分析甘蓝型油菜中BnKCS基因在根、茎、叶、花蕾、柱头、花粉、30d荚果皮、40d荚果皮、30d种子和40d种子中的表达量。各BnKCS基因在甘蓝型油菜不同组织或器官中具有不同的表达模式，暗示各个基因在不同的组织或器官行使功能。所分析的18个BnKCS基因都在油菜花器官中表达，10个BnKCS基因在茎中表达，13个基因在叶中表达，9个基因在种子中有表达，8个基因在角果皮中表达。BnKCS2、BnKCS7、BnKCS8、BnKCS9和BnKCS215个基因在花器官中特异表达，其中BnKCS2和BnKCS9只在花粉中特异性表达，BnKCS21仅在花瓣中特异表达。
将克隆的17个BnKCS基因构建到酵母表达载体pYES2/NTA中半乳糖诱导启动子的下游，在酵母中进行异源表达分析。15个BnKCS基因在酵母中正常表达并翻译蛋白，BnKCS3和BnKCS4受密码子偏好的影响在酵母中没有表达产物。分析酵母脂肪酸组成发现，只有转BnKCS1基因和FAE1基因的酵母中有超长链脂肪酸的合成，其它转基因酵母均没有新的超长链脂肪酸合成。结果表明这些BnKCS基因不能利用酵母中的脂肪酸作为催化底物。在后续研究中需要在酵母体外添加超长链脂肪酸做底物，进一步研究BnKCS基因的功能。
为了揭示KCS基因与蜡质合成的相关性，鉴定出了7个有突变体纯系的拟南芥kcs突变体，包括kcs4、kcs7、kcs9、kcs11、kcs13、kcs16和kcs17。基因表达谱分析显示，突变体中的靶标基因因T-DNA的插入而沉默。用扫描电镜扫描拟南芥突变体及野生型角果和茎表面的蜡质晶体密度，突变体与野生型相比，茎和角果表面的蜡质晶体密度没有明显变化。通过GC-MS分析kcs突变体角果、茎和叶的表面蜡质组成。发现除kcs4突变体外，其余的kcs突变体茎、角果和叶的部分蜡质组分与野生型相比有显著降低，且链长不一，既有酰基还原途径的产物，如初级醇，也有脱羰基途径的产物如烷烃、醛和29酮。kcs突变体没有出现出蜡质严重缺失的性状，也没出现某一链长的超长链脂肪酸衍生物过度积累或某一蜡质组分完全缺失的情况。表明本研究中的KCS7、KCS8、KCS9、KCS11、KCS13、KCS16和KCS17基因的催化功能与其他KCS基因相互重叠，催化产物不是某一特定链长的超长链脂肪酸，而且催化产物既参与酰基还原途径也参与脱羰基途径的代谢。
KCS gene family encode β-ketoacyl CoA synthase, a rate-limiting enzyme in the synthesis of very long chain fatty acid (VLCFA), catalyzing the condensation reaction of VLCFA elongation process. Cloning of KCSgenes help to elucidate the function of the KCSgenes in the synthesis of VLCFAs and wax, and provide a new source of genes for improving rapeseed fatty acid composition.
In this study, FAE1gene sequences were isolated from high erucic acid B. napus varieties Zhongyou821, zero-erucic acid wild species Orychophragmus violaceus L.and Capsella bursapastrois L., wild species containing erucic acid Sinapis alba L., Sinapis arvensis L. and Isatis indigotica Fort. Comparison of FAE1sequences indicated that the FAE1gene of Zhongyou821showed more than85%identity with that of five wild species. Heterologous expression of the FAE1genes was performed in the yeast cell, all the FAE1genes from the wild cruciferous species can normally express in yeast cells. The yeast cells expressing FAE1genes from O. violaceus and C. buraspastroil showed no VLCFA formation, and those expressing FAE1genes from S. arvensis, S. alba, and I. indigotica produced trace amount VLCFA. Experimental results show that the low erucic acid trait of O. violaceus and C. buraspastroil comes from the inactivation of protein activity encoded by FAE1gene. The FAE1nucleotide sequences are highly conserved between high erucic acid species and zero erucic acid species.
FAE1gene is the rate-limiting enzyme in the synthesis of erucic acid. The high erucic acid rapeseed varieties can be improved for low erucic acid rapeseed varieties through directed inhibition of FAE1gene function. The full-length sequence of FAE1gene was chosen to construct the FAE1RNAi vector. The FAE1sequences were cloned to downstream of the seed-specific Napin promoter as a head-to-head concatemer, and the inverted repeats of FAE1sequence were spaced apart by a histone H2A intron. The FAE1RNAi vector was transformed into high erucic acid B. napus varieties Zhongyou821by Agrobacterium-mediated method. After transformation,81transformants were obtained, in which,51positive seedlings were identified by PCR method. GC analysis of fatty acid composition of transgenic T1generation seeds revealed that the erucic acid content of transgenic rapeseed decreased in different degree compared with the wild type. The erucic acid content of recipient varieties Zhongyou821was43.35%, that of21transformants is less than35%, in which, the erucic acid content of two transformants decrease to10-20%. Near-infrared analysis of T2generation seeds showed that the erucic acid content of most lines displayed no significant difference compared with high erucic acid rapeseed. The erucic acid trait displayed separation ratio of1:3for the descendants of one line, the erucic acid content of low erucic acid transgenic plant was at the level of2%. The results revealed that the FAE1silence mediated by RNAi technology can effectively reduce the erucic acid content of transgenic plants, but can not reduce to zero erucic acid level. And low erucic acid transformants have obvious unintended effects, showing seed shrinkage, irregular shape, seed coat glossless and difficult to normally grow.
On the basis of previous genomewalking and RACE experiment, we cloned17KCS gene members, including BnKCS1, BnKCS3, BnKCS4, BnKCS5, BnKCS6, BnKCS8, BnKCS9, BnKCS10, BnKCS11, BnKCS12, BnKCS13, BnKCS15, BnKCS16, BnKCS17, BnKCSl9, BnKCS20and BnKCS21. According to the pedigree analysis, BnKCS gene members can be divided into six sub-categories. The sequence homology ranges from45.6%to63.1%between the FAE1gene and other members of the KCS gene family. BnKCS4, BnKCS8, BnKCS9, BnKCS16, BnKCS17and FAE1genes belong to one sub-category, these genes show more than60%identity with FAE1gene. Expression profile analysis of BnKCS genes showed that there are9BnKCS genes expressing in the seeds, including BnKCS1, BnKCS4, BnKCSS, BnKCS6, BnKCS9, BnKCS10, BnKCS11, BnKCS19and BnKCS20. these genes exhibit greater than50%homology with FAE1gene. It was speculated that the FAE1full-length sequence was used to construct FAE1RNAi vector, which not only inhibit the expression of FAE1gene, but also synergisticlly inhibit the expression of homologous genes, thus, resulting in unintended effects. In the future, it is better to identify FAE1specific sequence for constructing FAE1RNAi or MicroRNAi vector on the basis of a comprehensive analysis of the sequences of the KCS gene family, to achieve the specific purpose of inhibiting the function of the FAE1gene.
The conserved domain FAEl-CUT1-RPPA of KCS family was usend to search the KCS sequences across the whole genomic sequence of B. napus, and40KCS sequences were obtained. Wherereas, no homologous sequence of KCS3, KCS7, KCS8, KCS11, KCS12, KCS16and KCS21were retrieved. In this study, BnKCS3, BnKCS8, BnKCS11, Bn KCS12, BnKCS16and BnKCS21were isolated form B. napus. It is concluded that the KCS family contains46gene members at least in B, napus.
The real-time PCR method was used to analyze the expression profile of BnKCS genes in different tissues of B. napus, including root, stem, leaf, bud, stigma, pollen,30d pod skin,40d pod skin,30d seeds and40d seed. The BnKCS genes have different expression pattern in different tissues or organs. The analysis results shows that18BnKCS genes are expressed in the floral organ of B. napus,10BnKCS genes expressed in the stem,13BnKCS genes expressed in leaves,9genes expressed in seed,8genes expressed in the pod skin.5genes (BnKCS2, BnKCS7, BnKCS8, BnKCS9and BnKCS21) are specifically expressed in floral organ, of which, BnKCS2and BnKCS9are specially expressed in the pollen, BnKCS21is specifically expressed in the petals.
17BnKCS genes were cloned to downstream of the galactose-inducible promoter of the yeast expression vector pYES2/NT, to perform heterologous expression in yeast cells.15BnKCS gene can normally translate protein, and the BnKCS3and the BnKCS4fail to be expressed due to the codon preference. GC analysis of yeast fatty acid composition indicated that the VLCFAs were only observed in yeast expressing the BnKCS1and FAE1genes, and no VLCFA synthesis in yeast expressing other BnKCS genes. It was concluded that these BnKCS genes can not take advantage of the fatty acids in yeast as a catalytic substrate to synthesize VLCFA. In follow-up studies, VLCFAs should be added to the yeast medium in vitro for further studying of BnKCS gene function.
To reveal the relationship between the KCS genes and wax biosynthesis, the homozygotes of7Arabidopsis Kcs mutant were identified, including kcs4, kcs7kcs9, kcsll kcs13, kcs16and kcs17. The KCS gene expression profile analysis showed that the target gene in the mutant was silenced due to the insertion of the T-DNA. The wax crystal density of the surface of Arabidopsis siliques and stems were scanned using a scanning electron microscope, and found that the wax crystal density of the mutant does not significantly change compared with that of the wild type. GC-MS was carried out to analyze the wax composition of Arabidopsis siliques, stems and leaves. Compared to the wild-type, part of the wax components were significantly reduced in stems, siliques and leaves for kcs mutants with except of the kcs4mutant. And the wax components with significant change have different chain length, including both the product of the acyl reduction pathway, such as primary alcohols, and that of decarbonylation pathway, such as alkanes, aldehydes and29C ketone. The kcs mutant did not appear serious deficiencies of surface wax, or excessive accumulation of a derivative of VLCFA and total loss of someone wax composition. This study revealed that the catalytic function was overlapping with other KCS gene for the KCS7, KCS8, KCS9, ACS11, KCS13, KCS16and KCS17genes, their catalytic product is not a VLCFA with specific chain length, involved in the metabolism of both acyl reduction pathway and decarbonylation pathway.
引文
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