甘蓝型油菜F5H基因的克隆及反义调控烟草木质素生物合成_英文_

Cloning of cDNA encoding F5H from Brassica napus and regulation of lignin biosynthesis by expressing antisense gene in transgenic tobacco

SHI Zhujuan, LIU Guihua, SHEN Jinxiong, WANG Hanzhong, YANG Xiangdong

National Center for Oil Crops Genetic Improvement, Oil Crops Research Institute of CAAS,

Wuhan 430062, China Email: shizhujuan@; zyzy@

Abstract

The whole coding sequences of F5H were obtained by RT-PCR from Brassica napus cv. Zhongshuang 9. All the sequences

had the conserved functional domains and showed high identity with those of Brassica napus cv. Westar. From the sequences analysis, a new member of F5H gene distinguished from the published members was found. The result suggested there were at

least 3 members of F5H gene in Brassica napus. Antisense gene expression binary vector was constructed by inserting one F5H

gene after the xylem-specific-expression promoter C4H from Arabidopsis thaliana. With Agrobacterium-mediated transformation, many independent T0 antisense F5H transgenic tobacco plants have been obtained. The growth conditions of the transgenic plants

and the wildtype control were identical before flowering. In the reproductive growth stage, the transgenic plants grew slowly and

were slender with less leaves, but there were no obvious differences in florescence and seeding. Thin transverse sections were cut

from the basal-stem of some plants in uniform growth with a freezing microtome for histochemical coloration. The results of Wiesner reaction and Maüle reaction indicated lignin distribution of transgenic plants changing. Most mature transgenic plant roots had less fibres and obvious taproot.

Key words: Brassica napus; Lignin; Ferulate 5-hydroxylase (F5H); RT-PCR; Agrobacterium-mediated transformation

Introduction

Brassica napus is a major oilseed crop grown worldwide. A natural lodging event resulted in a yield reduction of 13-20% and more sensitive to pathogen infect (Goodman et al., 2001). Lignin is crucial for structural integrity of the cell wall and strength of the stem, and therefore plays a role in protecting plants against lodging and pathogens (Boerjan et al., 2003).

In dicotyledonous angiosperms, lignin is composed of two major monomeric phenolic constituents: guaiacyl (G) and syringyl (S) units. Increased G content leads to more highly condensed lignin composed of a greater proportion of biphenyl and other carbon-carbon linkages, whereas S subunits are commonly linked through more labile ether bonds at the 4-hydroxyl position (Li et al., 2000). Softwood gymnosperm lignin essentially lack S residue and therefore are highly condensed.

Ferulate 5-hydroxylase (F5H) is a rate-limiting enzyme determining syringyl-substitued lignin precursors (John et al., 2004). Earlier studies have demonstrated that lignin of the F5H mutant lacks the sinapic acid-derived components typical of wild-type lignin (Chapple et al., 1992), while over-expression of F5H increases lignin syringyl monomer content (Meyer et al., 1998; Franke et al., 2000). We intend to acquire more condensed lignin to impart dramatic resistance against lodging and pathogen by down-regulation of F5H activity. Here we report regulation of lignin biosynthesis by expressing antisense F5H gene under the control of the lignification-associated cinnamate-4-hydroxylase promoter in tobacco.

Materials and Methods

Plant material

Leaf and petiole tissues were collected from Brassica napus cv. Zhongshuang No.9 planted in Oil Crops Research Institute, Chinese Academy of Agricultural Sciences.

Tobacco was Nicotiana benthamiana, planted in chamber under 16-h light/25°C and 8-h dark/23°C cycle.

Cloning of cDNA encoding F5H from Brassica napus

Gene specific and degenerate primers were designed on the basis of comparing the published plant F5H sequences (Brassica napus: AF214007, AF214008, AF214009; Arabidopsis thaliana: At4g36220; Eucalyptus gunnii: AJ249093; Liquidambar styraciflua: AF139532), primarily referred to the sequences of Brassica napus and Arabidopsis thaliana. The primers are as following:

The forward primer: 5′-ATGGAGTCTTCTATATCACAAACACTAAG-3′;

The reverse primer: 5′-TTA(a,g)A(c,g)AG(a,c)ACA(a,g)AT(a,c)AGGCG(t,c)GTG-3′.

Vector construction and tobacco transformation

The plant expression vector was pBI121, which carried an nptII gene as a selection marker, the 35S promoter was substituted with Arabidopsis thaliana C4H promoter provided by Dr. Yang. The sequence encoding GUS of pBI121 was replaced by the F5H sequence in reverse orientation. The recombined plasmid was transformed into Agrobacterium tumefaciens LBA4404 by freezing-thawing methods. Nicotiana benthamiana tissue was transformed and regenerated into

whole plants as common methods.

Another primer corresponding to the NOS terminator region were used for detecting the regenerate tobaccos, that is: 5′-GATTGCTTTGATATTGTCACGGGT-3′.

Analysis of transgenic tobacco

In the reproductive growth stage, the lower stems of some plants in uniform growth were cut to 20µm transverse sections with freezing microtome for histochemical coloration. For Wiesner staining, sections were stained with 2% (w/v)

phloroglucinol (dissolved in 95% alcohol) in 12% HCl. For Maüle staining, sections were immersed in 1% (w/v) potassium permanganate solution for 5 min at RT, then washed twice with 3% hydrochloric acid until the color turned from black or dark brown to light brown.

Results and Discussion

Sequence analysis of the cDNAs encoding F5H

The whole coding sequences were obtained by RT-PCR from Brassica napus cv. Zhongshuang 9. All the deduced amino acid sequences of the F5H clones contained the motifs known to be conserved (Meyer et al., 1996). Immediately following the inferred initiator methionine was a 17-amino acid sequence containing nine hydroxy amino acids. The subsequent 15-amino acid sequence was rich in hydrophobic amino acids. An RRRR putative stop transfer sequence followed the hydrophobic stretch immediately. Following the stop transfer sequence was the sequence PPGPRGWP, which obeyed the consensus for the proline-rich sequence found in many P450s. The most obvious of these conserved regions was the heme-binding domain between residues Pro-450 and Gly-460, including Cys-458, the presumed heme-binding cysteine.

The clones could be classified to 3 groups. The sequences in one group showed over 99% identity each other at protein level (Nuances of amino acids were some similar amino acids replacements), so randomly choose one clone each group to analyze at protein level. The NO.1 clone only showed 94% identity with the NO.2 as those of Brassica napus cv. Westar (Remesh et al., 2000). The NO.1 clone contained a 1563 bp open reading frame coding 520 amino acids, while the NO.2 and the NO.3 both contained a 1560 bp open reading frame coding 519 amino acids. The similarity scores of the NO.3 clone with the other two were all 96%. The result suggested there were at least 3 members of F5H gene in Brassica napus. So a new member of F5H gene might be found in this study.

Antisense plant expression binary vector was constructed by inserting the NO.1 clone after xylem-specific-expression promoter C4H from Arabidopsis thaliana. The C4H promoter sequence was identity with the sequence reported by Mizutani M et al (1997), contained three cis-acting elements (box P, YTYYMMCMAMCMMC; box A, CCGTCC; and box L, YCYYACCWACC), which were conserved among the genes involved in the core reactions of the phenylpropanoid pathway of several plant species. These elements might be involved in coordinating C4H gene expression with regulation of the PAL and 4CL in response to wounding and light.

Analysis of antisense-F5H-transgenic tobacco

With Agrobacterium-mediated transformation, many independent T0 antisense F5H transgenic tobacco plants had been obtained. The growth of the transgenic plants and the wildtype control were almost identical before flowering. In the reproductive growth stage, the transgenic plants grew slowly and were slender with less leaves, but there were no obvious differences in florescence and seeding.

In the reproductive growth stage, thin transverse sections were cut for histochemical coloration (as showed in Fig 1). Wiesner reaction is the method to detect the total extent of lignin in the rough through shade of color, and Maüle reaction is the way to determine the content of S lignin (presented in red). In Wiesner reaction, the vascular bundle of some transgenic plants presented different shade of mauve strip, the vascular bundle near the marrow of some other transgenic plants showed visible deep mauve. However, the vascular bundle of the wildtype control were with even mauve. In Maüle reaction, the vascular bundle of some transgenic plants exhibited red strips and inter-phase yellow strips, the vascular bundle near the marrow of some other transgenic plants showed visible deep brown yellow and the periphery of the vascular bundle showed visible deep red, while the vascular bundle of the wildtype control were yellow changing to red from the periphery to the middle part gradually.

Most mature transgenic plant roots were with less fibres and obvious taproot than the control (Fig 2).

The F5H mutants exhibit a characteristic red fluorescence under UV, whereas wild-type plants have a blue-green appearance (Chapple et al., 1992). In the present study, all the T0 antisense F5H transgenic tobacco plants were not different from the wildtype in any growth stage to UV. This indicated the F5H activity existed in the transgenic tobacco plant. Referrences

1. Boerjan W, Ralph J, Baucher M. 2003. Lignin Biosynthesis. Annals Reviews of Plant Biol, 54: 519–549.

2. Chapple C C, Vogt T, et al. An Arabidopsis mutant defective in the general phenyl -propanoid pathway. Plant Cell, 1992, 4: 1413-1424.

3. Franke R, Mcmichael C M, Meyer K, et al. Modified lignin in tobacco and poplar plants over-expressing the Arabidopsis gene encoding ferulate

5-hydroxylase. Plant J, 2000, 22: 223-234.

4. Goodman A.M, Crook M.J, Ennos A.R. Anchorage Mechanics of the Tap Root System of Winter-sown Oilseed Rape. Annals of Bot, 2001, 87: 397-404.

5. Li L, Popko JL, Umezawa T, et al. 5-Hydroxyconiferyl aldehyde modulates enzymatic methylation for syringyl monolignol formation, a new view of

monolignol biosynthesis in angiosperms. J Biol Chem, 2000, 275: 6537–6545.

6. Meyer K, Cusumano,J.C., Somerville,C., et al. Ferulate 5-hydroxylase from Arabidopsis thaliana defines a new family of cytochrome P450-dependent

monooxygenases. Proc Natl Acad Sci, 1996, 93: 6869–6874.

7. Meyer K, Shirley AM, Cusumano JC, et al. Lignin monomer composition is determined by the expression of a cytochrome P450-dependent monooxygenase

in Arabidopsis. Proc Natl Acad Sci, 1998, 95: 6619–6623.

8. Mizutani M, Ohta D, Sato R. Isolation of a cDNA and a genomic clone encoding cinnamate 4-hydroxylase from arabidopsis and its expression manner in

planta. Plant Physiol, 1997, 113: 755-763.

9. Ramesh BN, Richard WJIV, Eugen K, et al. Identification of a CYP84 Family of Cytochrome P450-Dependent Mono-Oxygenase Genes in Brassica napus

and Perturbation of Their Expression for Engineering Sinapine Reduction in the Seeds. Plant Physiol, 2000, 123: 1623-1634.

ww mw

w1 m1

w2 m2

2

t2

Fig 1. Histochemical coloration of trangenic and untransformed tobaccos

ww: untransformed tobaccos in Wiesner reaction; w1 and w2: trangenic tobaccos in Wiesner reaction;

mw: untransformed tobaccos in Wiesner reaction; mi and m2: transgenic tobaccos in Maüle reaction

Fig 2. Root difference between transgenic tobaccos and untransformed tobaccos

The plants with tally are the transgenic tobacco plantlets, others are the CK.

114 生物技术:基因组学及其应用——BP-1-25、BP-1-26 甘蓝型油菜F5H基因的克隆及反义调控烟草木质素生物合成

师竹娟 刘贵华 沈金雄 王汉中 杨向东

中国农业科学院油料作物研究所/国家油料作物改良中心, 武汉 430062, 中国

Email: zyzy@

RT-PCR扩增得到油菜F5H基因完整编码区段，与已发表F5H基因序列高度同源，所翻译的氨基酸序列包含相应功能保守域。多个克隆测序，发现一个与已发表F5H基因序列存在较大差异的新家族成员，表明油菜F5H基因的基因家族至少存在3个成员。采用主要在木质部特异表达的拟南芥C4H启动子，以其中一个F5H基因cDNA为插入片段构建植物反义表达载体。叶盘法转化烟草，获得多个T0代转反义F5H基因株系。开花前，转基因烟草与空白对照生长发育情况大致相同。进入生殖生长阶段后，转基因烟草株系的茎杆较细，叶片较小，总体长势单薄，但开花结实并无明显差异；选择生长发育情况一致的烟草植株茎干基部，冰冻超薄切片，进行组织化学染色。Wiesner反应染色的深浅可粗略反应木质化部位的木质素总含量，而Maüle反应使S木质素呈现红色，G木质素呈现黄色，染色的深浅可粗略反应其含量。Wiesner反应中，转基因烟草株系总体表现为两类，一类维管束呈深浅不同的紫红条带，另一类在临近髓的维管束处呈明显加深的紫红，而空白对照的维管束为均一的紫红。Maüle反应中，转基因烟草株系总体表现为两类，一类维管束呈深浅不同的黄色条带，维管束外围略显红色，另一类在临近髓的维管束处呈明显加深的棕黄色，维管束外围呈明显加深的红色，而空白对照为维管束从外至内逐渐由黄转红。Wiesner反应和Maüle反应的结果初步说明，茎秆木质素的沉淀分布发生了变化。收获种子后挖掘根系，绝大多数转基因烟草株系表现为须根发育极少而有明显的主根，空白对照为繁茂的须根而无明显的主根。

低芥子碱油菜的选育

Juliane Mittasch Diana Schmidt Dieter Strack Carsten Milkowski

Department of Secondary Metabolism, Leibniz Institute of Plant Biochemistry, Weinberg 3, 06120 Halle, Germany

Email:cmilkows@ipb-halle.de

油菜籽粒中富集着大量影响营养价值的芥子碱脂，其主要以芥子碱的形式存在。因为这些酚类化合物味觉苦涩、容易和蛋白质形成复合物从而影响种子的加工生产性能，因此，加工后不仅仅影响菜籽饼粕饲料的适口性而且也阻碍了油菜粒蛋白质的食用食品市场化进程。因此，降低芥子碱含量是油菜作为蛋白食物的实质要求。芥子碱的生物合成途径包括两步：首先是在尿苷二磷酸-葡糖苷酸转移酶(SGT; UGT84A9)作用下芥子酸脂转化为芥子酸葡糖苷，然后芥子酸葡糖苷在 SCT催化条件下合成芥子碱。通过RNAi技术将UGT84A9 (BnSGT1)沉默后，种子中的SE含量显著下降。本文研究了芥子酸脂合成途径中相关基因在种子发育时期的分子特征及其等位基因、以及抑制代谢途径的方法。通过对BAC文库的筛选，发现甘蓝型油菜中UGT84A9至少有两个拷贝。种子中的UGT84A9蛋白活性主要是由来自于C基因组的SE相关的等位基因表达。通过EST克隆和候选基因法，发现了与UGT84A9相关的、可能与芥子酸葡糖苷合成相关的基因。这包括：分离到了编码羟基肉桂酸葡(萄)糖基转移酶(UGT84A10)的cDNA序列和13个在种子中表达且与尿苷二磷酸-葡(萄)糖基转移酶同源的EST序列。同时基于cDNA的克隆，还鉴定了编码BnSCT1的基因。来自A基因组和C基因组的BnSCT1基因的等位基因的特征得以阐明，它们都在种子发育过程中表达。启动子分析和RNA丰度实验证实了BnSCT1在种子中特异表达。拟南芥中该基因的沉默导致了芥子胆碱含量的降低，并且种子中芥子碱葡萄糖苷含量增加。为了有效地抑制油菜中芥子胆碱的合成，构建了UGT84A9 与 BnSCT1融合载体的用于RNA干涉，通过转基因得到了稳定的低芥子碱油菜。

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