滨麦低分子量谷蛋白亚基（LMW-GS）基因的分离与序列分析

采用PCR方法,从滨麦(Leymus mollis)基因组中分离出8条LMW-GS基因序列.核苷酸序列分析表明,序列GQ169791在起始密码子上游包含318 bp的启动子序列,该序列包含-300元件、GCN4 motif、种子贮藏蛋白盒等基因特异表达的顺式或反式作用调控元件.推导的氨基酸序列分析表明,8条序列的编码区依次有信号肽,N-末端区,中部重复区和C-末端Ⅰ、Ⅱ、Ⅲ区等典型LMW-GS多肽一级结构特征;序列HQ416909、HQ416914和HQ416915具有单一完整的开放阅读框(ORF);序列GQ169791、HQ416910、HQ416911、HQ416912和HQ416913在中部重复区和C-末端区出现了4个或5个提前终止密码子,推断其为假基因.8条序列都含有8个或9个半胱氨酸残基(C),N-末端区起始氨基酸序列为METSRIPG-或METTRIPG-,推断其为LMW-m型LMW-GS基因.系统进化分析表明,8条序列与华山新麦草(Psathyrostachys huashanica)LMW-GS基因(HM475146,GQ223386)和野大麦(Hordeum brevisubulatum)的B-hordein基因(AY695368)具有相对较近的同源关系.该研究为挖掘利用滨麦LMW-GS的基因提供了理论依据,对小麦品质改良具有一定参考价值.     

普通小麦低分子量麦谷蛋白亚基核心编码区与面团强度关系的研究

为分析LMW-GS基因对面团强度的影响, 利用两个重组自交系99G45/京771和Pm97034/京771的F9代,对LMW-GS基因特异位点和与其紧密连锁的Gli-1位点进行分析, 研究对面团强度影响差异显著的Glu-B3位点的等位基因核心编码区的序列差异。结果表明, 3个亲本LMW-GS核心编码区都具有6个半胱氨酸残基, 但PB较GB和JB缺失了一个7氨基酸序列的重复单元, 并且在不同序列中出现了氨基酸代换, 其中有2个代换可能影响氨基酸序列的亲水性, 进而影响面团强度。     

小麦Glu-B3位点LMW-GS基因的克隆及序列分析

以小麦特殊遗传材料———六倍体普通小麦阿勃二体、1A缺体、1B缺体和1D缺体,四倍体硬粒小麦墨西粒卡以及二倍体节节麦的总基因组DNA为模板,对D Ovidio等曾报道的硬粒小麦Glu-B3位点LMW-GS基因特异引物对P1(5-′tcctgagaagtgcatgacatg-3′)和P2(5-′gtaggcaccaactccggtgc-3′)进行了PCR扩增验证.结果表明,该引物对同样能特异扩增普通小麦Glu-B3位点LMW-GS基因.利用这对引物通过AS-PCR方法克隆得到优良小麦品种小偃6号1B染色体1个LMW-GS基因片段.该基因全长为1 089 bp,包含了完整的编码区和其上游318 bp的胚乳特异表达启动子区.该基因被命名为XY-Glu-B3-LMW2(GenBank登录号为DQ630442).XY-Glu-B3-LMW2的推测蛋白含256个氨基酸(包括N-端20个氨基酸的信号肽),其成熟蛋白有8个保守的Cys残基,均分布在C-末端区.XY-Glu-B3-LMW2是从小偃6号克隆到的第2个LMW-GS基因.     

小麦低分子量谷蛋白亚基基因5′侧翼保守序列染色体组特异性DNA变异的分析及验证

根据33个低分子量麦谷蛋白亚基(LMW-GS)基因5′侧翼序列的相似性进行聚类分析, 可将其划分成8个类群, 这与基于N末端推导氨基酸序列进行的类群划分结果完全一致. 序列比对发现, 各类群基因5′侧翼保守序列间存在DNA多态性, 共发现34个多态性位点, 其中18个为潜在单核苷酸多态性位点(SNPs, single nucleotide polymorphisms). 除1个LMW-GS类群之外, 其余7个类群的5′侧翼序列均具有类群特异性DNA变异位点. 根据类群间的DNA多态性对这7个类群设计了特异引物, 利用普通小麦(Triticum aestivum L.)品种中国春及其第1同源群双端体系对其进行染色体定位分析, 揭示了1AS, 1BS和1DS上分别有第2, 1和4类群. 对PCR产物的克隆测序进一步验证了不同染色体组上的LMW-GS基因类群间5′侧翼序列具有特异性. 这些结果表明, LMW-GS基因的编码区及其5′侧翼保守序列可能是协调进化的. 本文报道的7对引物可对7类LMW-GS基因的完整编码区进行特异扩增, 因而能在小麦复杂的遗传背景下有目的地对某一类LMW-GS基因进行分离克隆, 这有助于弄清单个LMW-GS对小麦品质的贡献. 同时, 在小麦育种中, 这些标记对于有效地选择与品质密切相关的LMW-GS组分有一定应用价值.     

雅致枝霉△6-脂肪酸脱氢酶基因的克隆及在酿酒酵母中的表达

根据真菌△^6 -脂肪酸脱氢酶基因保守的组氨酸Ⅱ区和Ⅲ区附近保守序列设计兼并引物进行RT-PCR,得到雅致枝霉（Thamnidium elegans）As3．2806△^6 -脂肪酸脱氢酶基因459bp部分cDNA序列,然后通过快速扩增cDNA末端技术（RACE）向两端延伸得到1504bp的△^6 -脂肪酸脱氢酶基因全长cDNA序列。序列分析表明有一个1377bp、编码459个氨基酸的开放阅读框TED6。推测的氨基酸序列与已知其他真菌的△^6 -脂肪酸脱氧酶基因的氨基酸序列比对,具有3个组氨酸保守区、2个疏水区及N末端细胞色素b5融合区。将此编码区序列亚克隆到酿酒酵母缺陷型菌株INVSel的表达载体pYES2.0中,构建表达载体pYTED6,并在酿酒酵母INVSel中异源表达。通过气相色谱（GC）和气相色谱,质谱（GC-MS）分析表明,该序列在酿酒酵母中获得表达,产生γ-亚麻酸（GLA）的含量占酵母总脂肪酸的7．5％。证明此序列编码的蛋白能将外加的亚油酸转化为γ-亚麻酸,是一个新的有功能的△^6 -脂肪酸脱氢酶基因（GenBank．AY941161）。     

重组链激酶及其三种突变体的活性研究

利用PCR技术从Streptococucspyogenes的基因组DNA中扩增了链激酶的编码基因ska,并进行了序列分析 ,利用基因删除及定点位变技术获得了删除了C-末端 42个氨基酸残基编码区的突变链激酶基因skaΔC42 ,第 5 9位Lys残基突变为Glu的突变链激酶基因skaK5 9E以及删除C-末端 42个氨基酸且第 5 9位Lys残基突变为Glu的突变链激酶基因skaΔC42K5 9E ,将ska及其三种突变体分别克隆到表达载体pET 1 5b上 ,构建分别表达野生型链激酶 (SK)、C-末端缺失 42个氨基酸残基的突变体 (SKΔC42 )、第 5 9位Lys残基突变为Glu的突变体 (SKK5 9E)及C-末端缺失 42个氨基酸且第 5 9位Lys残基突变为Glu突变体 (SKΔC42K5 9E)的表达载体pSK ,pSKΔC42 ,pSK K5 9E ,pSKΔC42K5 9E ,分别转化E .coliBL2 1 (DE3) ,IPTG诱导后在大肠杆菌中实现了高效表达 ,经亲和层析、离子交换层析及分子筛层析 ,获得了rSK、rSKΔC42、rSKK5 9E及rSKΔC42K5 9E ,活性分析表明rSK与其三种突变体具有相同的比活性。     

雅致枝霉Δ6-脂肪酸脱氢酶基因的克隆及在酿酒酵母中的表达

根据真菌Δ6-脂肪酸脱氢酶基因保守的组氨酸Ⅱ区和Ⅲ区附近保守序列设计兼并引物进行RT-PCR,得到雅致枝霉(Thamnidium elegans)As3.2806Δ6-脂肪酸脱氢酶基因459bp部分cDNA序列,然后通过快速扩增cDNA末端技术(RACE)向两端延伸得到1504bp的Δ6-脂肪酸脱氢酶基因全长cDNA序列。序列分析表明有一个1377bp、编码459个氨基酸的开放阅读框TED6。推测的氨基酸序列与已知其他真菌的Δ6-脂肪酸脱氢酶基因的氨基酸序列比对,具有3个组氨酸保守区、2个疏水区及N末端细胞色素b5融合区。将此编码区序列亚克隆到酿酒酵母缺陷型菌株INVSc1的表达载体pYES2.0中,构建表达载体pYTED6,并在酿酒酵母INVSc1中异源表达。通过气相色谱(GC)和气相色谱/质谱(GC-MS)分析表明,该序列在酿酒酵母中获得表达,产生γ-亚麻酸(GLA)的含量占酵母总脂肪酸的7.5%。证明此序列编码的蛋白能将外加的亚油酸转化为γ-亚麻酸,是一个新的有功能的Δ6-脂肪酸脱氢酶基因(GenBank,AY941161)。     

猪I-FABP基因的分子克隆与组织特异性表达分析

小肠型脂肪酸结合蛋白对长链脂肪酸具有高度的亲和力,参与脂肪酸的吸收和细胞内转运。利用cDNA末端快速扩增（RACE）技术并结合同源克隆策略,克隆到了编码猪小肠型脂肪酸结合蛋白基因（I-FABP）的全长cDNA序列（GenBank接受号：AY960624）,并对系统发育关系等进行了生物信息学分析。猪I-FABP基因的cDNA序列全长614 bp,其中包括399bp的开放式读码框（ORF）,43bp的5’末端非编码区（5’URT）和172bp的3’末端非编码区（3’URT）,编码132个氨基酸残基蛋白,在氨基酸水半上与其他物种的I-FABP具有高度的同源性。以邻接法（Neigbor-Joining,NJ）所构建的系统发育关系表明,猪I-FABP与其他物种的,I-FABP属于同一类群,且与人的遗传距离最近。Northern杂交和半定量RT—PCR分析发现,猪I-FABP在猪体组织中出现约620bp大小的转录本,且在猪体组织中广泛存在,但在小肠组织中表达量最为丰富。     

西伯利亚PsPIP1基因的克隆及其在NaHCO3胁迫下的表达

应用cDNA末端快速扩增（RACE）技术从西伯利亚蓼叶cDNA文库中克隆了质膜内在蛋白基因（PsPIP1的完整编码区cDNA序列（GenBank accession No．EU626398）,长度为1004bp,编码285个氨基酸。基于和其他植物水通道蛋白的氨基酸序列、推测的三维结构的比较以及系统进化分析结果,初步确定此基因为水通道蛋白基因家族中的PIP1亚族成员。RT-PCR结果显示,PsPIP1在西伯利亚蓼的地下茎、茎、叶中均有表达,叶中表达量最高,地下茎次之,茎中最低。在NaHCO3胁迫与去胁迫的过程中,此基因在地下茎、茎、叶中的表达模式也有较明显的差异。     

犬黑素皮质素受体-2基因的分子克隆及序列分析

目的分离犬MC2R基因cDNA5′末端,分析其启动区域特点。方法采用了RNA连接酶介导的RACE（RLM-RACE）技术分离了犬MC2R基因和局部序列比对工具（Basic Local Alignment Search Tool,BLAST）对CDS区进行了初步验证。结果新分离了犬MC2RcDNA的5′末端,并对其启动区序列作了初步分析。序列分析显示,该基因至少由两个外显子（exon1和exon2）组成,exon1和exon2的一部分编码5′非翻译区（5′-UTR）,exon2其余的部分编码整个编码区。结论克隆了犬MC2R基因的5′末端,在其启动区发现了inr、SF-1、SP1、CRE、PPRE、AP-1等多个顺式作用元件,为犬MC2R表达调控研究奠定基础。     

Characterization of a Low-Molecular-Weight Glutenin Subunit Gene from Bread Wheat and the Corresponding Protein That Represents a Major Subunit of the Glutenin Polymer

Both high- and low-molecular-weight glutenin subunits (LMW-GS) play the major role in determining the viscoelastic properties of wheat (Triticum aestivum L.) flour. To date there has been no clear correspondence between the amino acid sequences of LMW-GS derived from DNA sequencing and those of actual LMW-GS present in the endosperm. We have characterized a particular LMW-GS from hexaploid bread wheat, a major component of the glutenin polymer, which we call the 42K LMW-GS, and have isolated and sequenced the putative corresponding gene. Extensive amino acid sequences obtained directly for this 42K LMW-GS indicate correspondence between this protein and the putative corresponding gene. This subunit did not show a cysteine (Cys) at position 5, in contrast to what has frequently been reported for nucleotide-based sequences of LMW-GS. This Cys has been replaced by one occurring in the repeated-sequence domain, leaving the total number of Cys residues in the molecule the same as in various other LMW-GS. On the basis of the deduced amino acid sequence and literature-based assignment of disulfide linkages, a computer-generated molecular model of the 42K subunit was constructed.     

四川小麦地方品种AS1643中低分子量谷蛋白基因的克隆及其蛋白质的二级结构预测

根据小麦低分子量谷蛋白基因保守区序列设计引物P1/P2, 采用PCR法对四川小麦地方品种AS1643的基因组DNA进行扩增, 获得1条约900 bp的片段, 分离、纯化后连接到载体pMD18-T上, 对筛选阳性克隆测序, 获得1个低分子量谷蛋白基因LMW-AS1643(GenBank登录号: EF190322), 其编码区长度为909 bp, 可编码302个氨基酸残基组成的成熟蛋白。序列分析结果表明, LMW-AS1643具有典型的低分子量谷蛋白基因的基本结构, 其推导氨基酸序列与其它已知的LMW-GS相比, 最高相似性为93.40%。生物信息学分析表明, 在LMW-AS1643低分子量谷蛋白中, 无规则卷曲含量最高, 为67.90 %, 其次是a-螺旋, 占30.46 %, b-折叠含量最少, 为1.64 %。     

Isolation of low-molecular-weight glutenin subunit genes from wild emmer wheat (Triticum dicoccoides)

Three low-molecular-weight glutenin subunit (LMW-GS) genes, designated LMW-Td1, LMW-Td2 and LMW-Td3, were isolated from wild emmer wheat (Triticum dicoccoides), which is the tetraploid progenitor of common wheat (T. aestivum). The complete nucleotide sequence lengths of LMW-Td1, LMW-Td2 and LMW-Td3 are 858, 900 and 1062 bp, respectively. LMW-Td1 and LMW-Td3 can encode proteins with 284 and 352 amino acid residues, respectively, whereas LMW-Td2 is a putative pseudogene due to the presence of 3 inframe stop codons in its C-terminal domain. The deduced protein sequences of the 3 genes share the same typical polypeptide structures with known LMW-GS genes containing 8 cysteines in the mature protein domains. LMW-Td1 was clearly distinguished from all known LMW-GS genes, and considered as a novel LMW-GS gene. Two hydrophobic motifs (i.e. PIIIL and PVIIL) were observed in the repetitive domain of LMW-Td3. Sequence comparison indicates that sequences of the 3 LMW-GS genes from this study are strongly similar to known LMW-GS genes. Our phylogenetic analysis suggests that LMW-Td1 and LMW-Td2 are homologous with genes on chromosome 1A, and LMW-Td3 is closely related to genes on chromosome 1B.     

Functional properties of a new low-molecular-weight glutenin subunit gene from a bread wheat cultivar

Some allelic forms of low-molecular-weight glutenin subunit (LMW-GS) can greatly influence the end-use of wheat flours, understanding the function of each allele of LMW-GS is important to wheat quality breeding. A LMW-GS gene XYGluD3-LMWGS 1(AY263369) has been cloned from bread wheat cultivar Xiaoyan 6. The deduced protein contained nine cystine residues, one more than that in all other LMW-GSs reported previously, indicating that it is either a new gene or a new allele of a known LMW-GS gene. In this study, the gene was expressed in E. coil in large scale for the testing of its functional property. Reactive Red 120-Agarose resin was used efficiently to purify the expressed LMW-GS proteins from bacteria, with the lactic acid–sodium lactate buffer (pH 4.5) which contained low concentration SDS as elution solution. The purified protein (belonging to the LMW-m family, MW about 35 KDa) was supplemented into a base flour, the results of 10 g dough mixing test indicated that incorporation of the LMW-GS increased the strength of the dough, with significant increases in mixing time (MT) and peak width (PW), and decrease in breakdown in resistance (RBD) compared with the control. In addition, the dough with incorporation of the LMW-GS had more glutenin macropolyeric protein than the control, suggesting that the LMW-GS participated in forming larger glutenin polymers, and greatly contributed to dough strength. The changes in mixing parameters and the amount of glutenin macropolyeric protein were related to the quantity of incorporating subunits.     

Molecular characterization of novel low-molecular-weight glutenin genes inAegilops longissima

Extensive genetic variations of low-molecular-weight glutenin subunits (LMW-GS) and their coding genes were found in the wild diploid A- and D-genome donors of common wheat. In this study, we reported the isolation and characterization of 8 novel LMW-GS genes fromAe.longissima Schweinf. & Muschl., a species of the sectionSitopsis of the genusAegilops, which is closely related to the B genome of common wheat. Based on the N-terminal domain sequences, the 8 genes were divided into 3 groups. A consensus alignment of the extremely conserved domains with known gene groups and the subsequent cluster analysis showed that 2 out of the 3 groups of LMW-GS genes were closely related to those from the B genome, and the remaining was related to those from A and D genomes of wheat andAe. tauschii. Using 3 sets of gene-group-specific primers, PCRs in diploid, tetraploid and hexaploid wheats andAe. tauschii failed to obtain the expected products, indicating that the 3 groups of LMW-GS genes obtained in this study were new members of LMW-GS multi-gene families. These results suggested that theSitopsis species of the genusAegilops with novel gene variations could be used as valuable gene resources of LMW-GS. The 3 sets of group-specific primers could be utilized as molecular markers to investigate the introgression of novel alien LMW-GS genes fromAe. longissima into wheat.     

Molecular characterization of LMW-GS genes from a somatic hybrid introgression line Ⅱ-12 between Triticum aestivum and Agropyron elongatum in relation to quick evolution

In order to exploit the evolution and find novel low-molecular-weight glutenin subunit(LMW-GS)for improvement of common wheat quality,thirteen variants from a somatic hybrid introgression line Ⅱ-12 between Triticum aestivum cv.Jinan 177(JN177)and Agropyron elongatum were characterized via genomic PCR.Four clones were pseudogenes because they contained an internal stop codon.The remaining nine variants contained intact open reading frames(ORFs).Sequence alignment indicates that the proteins deduced from the nine ORFs have similar primary structure with LMW-GS cloned from its parents previously.However,they have some unique modifications in the structures.For example,EU292737 contains not only an extra Cys residue in the C-terminal domain but also a long repetitive domain.Both EU159511 and EU292738 start their first Cys residue in the N-terminal repetitive domain,but not in the N-conserved domain traditionally.These structural alterations may have positive contributions to wheat flour quality.The results of phylogeny showed that most LMW-GS variances from Ⅱ-12 were homologous to those from parent JN 177 and other wheat lines.The reason for quick evolution of LMW-GS in Ⅱ-12 was discussed.     

Structure,variation and expression analysis of glutenin gene promoters from Triticum aestivum cultivar Chinese Spring shows the distal region of promoter 1Bx7 is key regulatory sequence

In this study, ten glutenin gene promoters were isolated from model wheat (Triticum aestivum L. cv. Chinese Spring) using a genomic PCR strategy with gene-specific primers. Six belonged to high-molecular-weight glutenin subunit (HMW-GS) gene promoters, and four to low-molecular-weight glutenin subunit (LMW-GS). Sequence lengths varied from 1361 to 2554 bp. We show that the glutenin gene promoter motifs are conserved in diverse sequences in this study, with HMW-GS and LMW-GS gene promoters characterized by distinct conserved motif combinations. Our findings show that HMW-GS promoters contain more functional motifs in the distal region of the glutenin gene promoter (> − 700 bp) compared with LMW-GS. The y-type HMW-GS gene promoters possess unique motifs including RY repeat and as-2 box compared to the x-type. We also identified important motifs in the distal region of HMW-GS gene promoters including the 5′-UTR Py-rich stretch motif and the as-2 box motif. We found that cis-acting elements in the distal region of promoter 1Bx7 enhanced the expression of HMW-GS gene 1Bx7. Taken together, these data support efforts in designing molecular breeding strategies aiming to improve wheat quality. Our results offer insight into the regulatory mechanisms of glutenin gene expression.     

Classification of wheat low-molecular-weight glutenin subunit genes and its chromosome assignment by developing LMW-GS group-specific primers

On the basis of sequence analysis, 69 known low-molecular-weight glutenin subunit (LMW-GS) genes were experimentally classified into nine groups by the deduced amino acid sequence of the highly conserved N-terminal domain. To clarify the chromosomal locations of these groups, 11 specific primer sets were designed to carry out polymerase chain reactions (PCR) with the genomic DNA of group 1 ditelosomic lines of Chinese Spring, among which nine primer sets proved to be LMW-GS group-specific. Each group of LMW-GS genes was specifically assigned on a single chromosome arm and hence to a specific locus. Therefore, these results provided the possibility to predict the chromosome location of a new LMW-GS gene based on its deduced N-terminal sequence. The validity of the classification was confirmed by the amplifications in 27 diploid wheat and Aegilops accessions. The length polymorphisms of LMW-GS genes of groups 1 and 2, and groups 3 and 4.1 were detected in diploid A-genome and S-genome accessions, respectively. The diploid wheat and Aegilops species could be used as valuable resources of novel allele variations of LMW-GS gene in the improvement of wheat quality. The nine LMW-GS group-specific primer sets could be utilized to select specific allele variations of LMW-GS genes in the marker-assisted breeding. Electronic Supplementary Material Supplementary material is available for this article at Hai Long and Yu-Ming Wei are the two authors who have contributed equally to this paper     

Comprehensive identification of LMW-GS genes and their protein products in a common wheat variety

Although it is well known that low-molecular-weight glutenin subunits (LMW-GS) from wheat affect bread and noodle processing quality, the function of specific LMW-GS proteins remains unclear. It is important to find the genes that correspond to individual LMW-GS proteins in order to understand the functions of specific proteins. The objective of this study was to link LMW-GS genes and haplotypes characterized using well known Glu-A3, Glu-B3, and Glu-D3 gene-specific primers to their protein products in a single wheat variety. A total of 36 LMW-GS genes and pseudogenes were amplified from the Korean cultivar Keumkang. These include 11 Glu-3 gene haplotypes, two from the Glu-A3 locus, two from the Glu-B3 locus, and seven from the Glu-D3 locus. To establish relationships between gene haplotypes and their protein products, a glutenin protein fraction was separated by two-dimensional gel electrophoresis (2-DGE) and 17 protein spots were analyzed by N-terminal amino acid sequencing and tandem mass spectrometry (MS/MS). LMW-GS proteins were identified that corresponded to all Glu-3 gene haplotypes except the pseudogenes. This is the first report of the comprehensive characterization of LMW-GS genes and their corresponding proteins in a single wheat cultivar. Our approach will be useful to understand the contributions of individual LMW-GS to the end-use quality of flour.     

Development of primers specific for LMW-GS genes located on chromosome 1D and molecular characterization of a gene from Glu-D3 complex locus in bread wheat

Glutenins are multimeric aggregates of high molecular weight (HMW) and low molecular weight (LMW) subunits, which determine the quality in wheat. Development of locus-specific primers is an important step toward cloning specific LMW glutenin subunits (LMW-GS) by PCR method. Based on the publicly available, a pair of primer, namely primer 3 (5' TTGTAGAAACTGCCATCCTT 3') and primer 4 (5' GTCACCGCTGCAT CGACATA 3') was designed and verified to specific for LMW-GS genes located on chromosome 1D in this study. The LMW-GS gene located at the Glu-D3 locus in bread wheat cultivar Xiaoyan 6 was cloned using this pair of primer. The clone designated as XYGluD3-LMWGS1 (AY263369), contains the endosperm-specific-expression promoter and the entire coding region. Nucleotide sequence comparison of the XYGluD3-LMWGS1 with other reported LMW-GS genes located at different Glu-3 loci showed the degree of identity among them ranged from 59.57% to 99.78%. The LMW-GS genes at the same locus showed more similar to each other than to the gene at different locus. Comparison of the deduced amino acid sequence of the XYGluD3-LMWGS1 with the sequences of 12 group LMW-GSs of wheat cultivar Norin 61 showed that the deduced amino acid sequence was nearly the same to LMW-GS group 10 (identity 99.67%). The deduced LMW-GS contains nine cystine residues, which contained one more cystine residue in the C-terminal conserved domain than previous reported. This was the first LMW-GS gene encoding for a LMW-GS with 9 cystine residues that has been discovered so far.