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Original Paper
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Acta Biochim Biophys
Sin 2007, 39: 377-383 |
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doi:10.1111/j.1745-7270.2007.00284.x |
Inheritance and Expression of
Copies of Transgenes 1Dx5 and 1Ax1 in Elite Wheat (Triticum
aestivum L.) Varieties Transferred from Transgenic Wheat through
Conventional Crossing
Sanhe LI, Ju LI, Nali WANG, Yuesheng
WANG, Guangxiao YANG, Jingye FANG, and Guangyuan HE*
China-UK
HUST-RRes Genetic Engineering and Genomics Joint Laboratory, Huazhong
University of Science and Technology, Wuhan 430074, China
Received: December
25, 2006�������
Accepted: February 25,
2007
This work was
supported by a grant from the Major State Basic Research Development Program of
China (No. 2002CB111302)
*Corresponding
author: Tel, 86-27-87792271; Fax, 86-27-87792272; E-mail,
[email protected]
Abstract������� To study the inheritance and expression of multiple copies of transgenes from transgenic wheat lines, three crosses between transgenic wheat lines B72-8-11b and B102-1-2 and Chinese elite wheat varieties Chuan89-107 and Emai18 were carried out. Chuan89-107�B72-8-11b, Chuan89-107�B102-1-2 and Emai18�B72-8-11b, and F1 plants were selfed or backcrossed to obtain different generation populations. Protein analysis in grains of F1 and F2 and backcross progenies of BC1F1, BC1F2, BC1F3, BC2F1, BC2F2 and BC2F3 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the transgenes 1Dx5 and 1Ax1 were expressed and segregated in the target wheat according to Mendelian laws. A range of 1Dx5 expression levels were observed in the progenies of Chuan89-107�B72-8-11b and Emai18�B72-8-11b, but the expression levels of 1Ax1 in progenies of Chuan89-107�B102-1-2 rarely changed. It suggested that the two foreign genes had different mechanisms of expression in the cross progeny, even though they were produced in the same way and the foreign 1Dx5 gene of 5-10 copies had the more complicated expression mechanism than the 1Ax1 gene of 4-5 copies.
Key words������� cross; elite wheat; 1Dx5 gene; 1Ax1 gene;
inheritance; expression
Wheat is the most important crop in the world in terms of its geographical distribution, area under cultivation and total yield. Ninety-five percent of cultivated wheat is of the hexaploid type used for the preparation of bread and other baked products. The types and quantities of high molecular weight glutenin subunits (HMW-GS) of wheat have a direct influence on the elasticity and strength of dough, which determine the bread-making quality [1-3].
Two HMW-GS genes are present on each of the homologous group one chromosomes of wheat, encoding an x-type and a y-type subunit [4]. Cultivars of hexaploid bread wheat containing three, four or five individual subunits (1Dx, 1Dy, 1Bx and 1Ax and/or 1By subunits) exist, but cultivars rarely contain six subunits because one or more of the six HMW-GS genes may be silenced. These differences in HMW subunit composition result in both quantitative and qualitative effects on bread-making performance in bread wheat [5,6]. Therefore, attempts to improve grain quality have focused on manipulating the amount and composition of the HMW-GS, especially 1Dx5 and 1Ax1 subunits which are known to be associated with good bread-making quality [4,7].
In China, most elite wheat varieties are not bread-making, and there is increasing demand for high quality dough for a range of food products. Good bread-making quality depends on the presence of protein subunit combinations such as 1Dx5+1Dyl0, 1Ax1, 1Ax2* and 1Bxl7+1Byl8 [8], but these are scarce in Chinese wheat. Only 30% of the varieties contain 1Dx5+1Dyl0 subunits compared to a higher percentage in the varieties in other countries [9].
Transgenic wheat line B72-8-11b contains 1Dx5, 1Bx17 and 1By18 subunits and line B102-1-2 contains 1Ax1, 1Bx17 and 1By18 subunits [10]. In these two wheat lines, the subunits they contain confer good wheat bread-making quality and they can offer multiple copies of the foreign gene. However, these two wheat lines lack other agricultural assets, for instance, they can not adapt well to the climate and environment in China and are not good in yield. The two elite varieties have good agricultural qualities such as high yield, resistance to some diseases, early ripening, and adaptation to the environment in Hubei province, but they might also have bread-making quality if they expressed the 1Dx5 or 1Ax1 gene. Crossing and backcrossing can combine the high HMW-GS in B72-8-11b and B102-1-2, with the assets of the elite varieties.
In the present study, crosses and backcrosses were made between transgenic lines and elite cultivars, Chuan89-107 and Emai18. Inheritance and expression of transgenes were analyzed with the aim of increasing the proportions of the HMW subunits and expression levels of foreign genes in Chinese elite wheat and improving its bread-making performance.
Materials and Methods
Plant materials
Transgenic lines B72-8-11b and B102-1-2 were used [10]. They were in the L88-31 background [11] and were produced by co-bombardment with the plasmid pAHC25 and either the p1Ax1 plasmid [5] or the p1Dx5 plasmid [12] containing the HMW subunit 1Ax1 and 1Dx5 genes, respectively, under the control of their own endosperm-specific promoters. B72-8-11b contains approximately 5-10 copies of the 1Dx5 gene and B102-1-2 contains approximately 4-5 copies of the 1Ax1 gene [10,13,14]. The two elite varieties are Chuan89-107 and Emai18, both with high yield and stress resistance.
All the wheat lines in this study were planted at the end of October or the beginning of November and were harvested the next May. The HMW-GS compositions of the materials used in this study are listed in Table 1.
Crossing and backcrossing
Three crossing and backcrossing combinations were carried out between Chuan89-107, Emai18 and B72-8-11b, B102-1-2: Chuan89-107�B72-8-11b, Chuan89-107�B102-1-2 and Emai18�B72-8-11b. Transgenic wheat lines were used as male parents and the other two lines were used as female parents and recurrent parents. Seeds of cross progenies F1, F2 and backcross progenies BC1F1, BC1F2, BC1F3, BC2F1, BC2F2 and BC2F3 were obtained from plants expressing the foreign gene in the previous generation.
Analysis of transgene
integration
For polymerase chain reaction (PCR) and Southern analysis, total genomic DNA was isolated from leaf tissue of wheat using the hexadecyltrimethylammonium bromide (CTAB) method [15]. PCR of the 1Ax1 and 1Dx5 genes was carried out with 50-200 ng of genomic DNA in a reaction mixture containing 50 mM KCl, 10 mM Tris-HCl (pH 8.8), 1.5 mM MgCl2, 0.1% Triton X-100, 200 mM each dNTP, 0.3 mM each primer, and 0.66 U of Taq DNA polymerase (TaKaRa, Dalian, China). The conditions for PCR of the 1Ax1 gene were one cycle of denaturation at 94 �C for 5 min, followed by 30 cycles of 94 �C for 30 s, annealing at 60 �C for 30 s, extension at 72 �C for 1 min, and a final extension at 72 �C for 10 min. The primers for the 1Ax1 gene were 5'-gtgtgagcgcgagct�cca�ggaa-3' and 5'-cggagaagttgggtagt�accctgc-3'. There was one difference in the 1Dx5 reactions, in that the extension phase was 72 �C for 2.5 min in the cycles. The primers for the 1Dx5 gene were 5'-gCCt�agc�AACcTTCAcAaTC-3' and 5'-gaAA�CCtgCT�gCGgA�cAAg-3'. Products of PCR amplification were analyzed by electrophoresis in 1.0% (W/V) agarose gels. Integration of foreign HMW subunit genes was examined by PCR and Southern blot analysis of digested genomic DNA. Genomic DNA was digested with EcoRI, which cut the plasmid pHMW1Ax1 into two parts, one of which was 7 kb long and includes the 1Ax1 gene, and the other was the 2.7 kb of plasmid puc8. EcoRI also cut the plasmid pHMW1Dx5 into two fragments of 8.7 kb and 2.7 kb. DNA was then separated by electrophoresis in 0.8% (W/V) agarose gel and transferred by capillary blotting to positively charged nylon membrane (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's instructions. Filters were hybridized with PCR-generated digoxigenin-labeled probes produced using a PCR digoxigenin probe synthesis kit (Roche Diagnostics).
Analysis of transgene
inheritance and expression
Seeds from each generation of the crossed and backcrossed wheat lines were germinated and grown in the field. Foreign gene segregation and inheritance were investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The distal, endosperm-containing parts of individual seeds were analyzed by SDS-PAGE for expression of the foreign HMW subunit genes. The corresponding proximal parts containing the embryo were then germinated, allowing transgene presence to be correlated with expression.
Total proteins of seeds were extracted from single half-grains in 25 ml/mg extraction solution of 62.5 mM Tris-HCl buffer, pH 6.8, containing 2% (W/V) SDS, 10% (V/V) glycerol and 0.02% (W/V) bromphenol blue, and separated by SDS-PAGE using a Tris-borate buffer system and 10% (W/V) polyacrylamide gels with approximately 0.09% ammonium persulfate (Sigma, St. Louis, USA) and 0.0008% tetramethylethylenediamine (TEMED) [16]. Acrylamide concentration in the stacking gel was 2%. The electrophoresis conditions were 130 V in the concentrating gel and 240 V in the separating gel. When the electrophoresis was finished, the gel was stained with a solution of 40% methanol, 10% trichloroacetic acid and 0.1% Coomassie Brilliant Blue R250.
Results
Analysis of crossing validity
To avoid false positives from crossing, SDS-PAGE was used to detect the expression of foreign genes of the positive seeds from the F1 generation. The results showed that the whole HMW-GS was correctly integrated and expressed (Fig. 1). Three combinations of crosses were also analyzed for their foreign HMW-GS genes by PCR. In the F1 generation, foreign genes could be amplified in all individual plants. The 1Ax1 gene was more than 400 bp, and the 1Dx5 gene was between 300 and 400 bp as shown on the agarose gel (Fig. 2). These results showed that the crosses were valid and the seeds could be used for breeding and backcrossing.
Analysis of foreign gene
integration
PCR analysis was also carried out in other generations. In the backcross progenies, foreign genes were segregated and absent from some plants of F2, BC1F2 and BC2F2. Southern blot was carried out and the results showed that there were still multiple copies of foreign genes in the offspring of the cross and backcross (data not shown).
HMW-GS expression and
segregation
The composition of HMW-GS was determined in the seeds of crossed and backcrossed progenies F2, BC1F2, BC1F3, BC2F1, BC2F2 and BC2F3. HMW-GS segregation was observed in the backcrossed progenies, and there were many genotypes (Fig. 3). There were some new HMW subunits which did not exist in the parent wheat lines [Figs. 3(A) and 4(B)].
Foreign HMW-GS expression
Foreign genes in transgenic wheat B72-8-11b and B102-1-2 were transferred by particle bombardment and the copy numbers of foreign genes were 5-10 for 1Dx5 and 4-5 for 1Ax1 [13,17]. Leonie [17] et al. found that 4-5 copies of 1Ax1 genes had been inserted at two loci in B102-1-2. Theoretically, when occurring at two loci, these 1Ax1 genes would segregate in the progenies of crosses and backcrosses, and the segregation ratios in F2 and BC1F1 were calculated, as shown in Table 2. Obviously, the observed ratios are close to the predictions of segregation ratios of two loci, but not for one locus.
Despite the possibility that the number of 1Ax1 genes varied, the expression levels of 1Ax1 were rarely changed [Fig. 4(A)].
In the chromosomes of wheat line B72-8-11b, 5-10 copies of foreign genes could be integrated at one locus or several loci. When considering only the foreign gene 1Dx5, the theoretical segregation ratio of BC2F1 would be 1:1, whereas the segregation ratios in F2, BC1F2 and BC2F2 generations would be 3:1 if the copies of the foreign gene were linked to form a single locus. A c2-test was carried out to test whether the foreign genes were inserted in one locus or two loci. The results were consistent with the predictions of one locus (Table 2). Results show that the multi-copy foreign genes in the transgenic wheat were inserted at one locus and they were inherited by the next generation according to Mendelian patterns.
However, the expression levels of the 1Dx5 gene were clearly different among the different progenies [Fig. 4(B,C)]. The expression of the 1Dx5 gene was at high levels in some plants but at low levels in others. Generally, the expression level of transgenes was higher than that of the endogenous 1Dx5 gene in L88-6, which was the isogenic wheat line of L88-31 containing one copy of the1Dx5 gene [Fig. 3(A)].
Discussion
Although particle bombardment has become the most popular method for the transformation of wheat, the disadvantages such as high copy number, unstable heritability and expression of the foreign gene were difficult to resolve. Embryos of many wheat lines have low embryogenic capacity, and the transformation frequency by particle bombardment is also very low (<1%). Therefore, it is not easy to obtain transgenic plants for these wheat lines in this way. Crossing and backcrossing between transgenic wheat and elite wheat can avoid these problems. It is easier to obtain lines with good desirable characters for many elite varieties using transgenic technology with conventional breeding than through the transgenic method of bombardment. There are many reports about the inheritance and stability of transgenes in donor wheat lines, and some research showed that once integration of foreign genes occurred, foreign DNA would be retained through meiosis and maintained in the progenies [9,14,18-20]. However, there are few reports about inheritance and expression of transgenes in elite wheat varieties. Only when foreign genes can be inherited stably by their progeny can this breeding mode be applied. The analysis of the inheritance and expression of copies of foreign genes in elite wheat became the goal of the present study.
In this work, crosses and backcrosses were used to study the inheritance and expression of multiple copies of the foreign genes 1Dx5 and 1Ax1 in the elite wheat lines whose genotypes are different from the foreign gene donors. The results showed that the multiple copies of foreign 1Dx5 and 1Ax1 genes could be inserted into the chromosomes of elite wheat lines during crossing and backcrossing, and these two genes were inherited by their progenies. The HMW-GS genes in these progenies segregated normally and there were various genotypes, some of which included the foreign genes, but the others did not.
If the foreign genes are inserted at a single locus, they will be linked and inherited together. In the course of inheritance, multiple copies of the same gene unite together and are inherited as a whole. Alternatively, the foreign gene insertion locus could be multiple and the foreign gene copy numbers are not always the same in each insertion locus. These foreign genes are dispersed randomly. It has been reported frequently that multiple foreign genes have been inserted in one locus during transformation by particle bombardment [18,21,22] and, in our trial, the foreign multiple copies of 1Dx5 gene were inserted in one locus and inherited as a whole.
Although the functional foreign 1Dx5 gene was inserted at a single locus, transformation by particle bombardment frequently results in foreign genes with high copy numbers. Some, but not all, of these foreign genes may be silenced because of gene rearrangement or co-suppression. Silenced genes are not expressed and therefore can not be observed by SDS-PGE. Non-silenced foreign genes showed various expression levels in the cross progenies of Chuan89-107�B72-8-11b and Emai18�B72-8-11b. 1Dx5 genes (5-10 copies) were inserted in one locus in the transgenic parent B72-8-11b. The expression levels of 1Dx5 in the cross progenies were sometimes lower than in the cross parent B72-8-11b and we can speculate that a proportion of multiple copies of a gene are silenced. Why the 1Dx5 gene was overexpressed stably in B72-8-11b but its expression level varied in the cross progenies is not clear.
Integration of 1Dx5 gene also led to the expression of an extra subunit whose molecular weight was similar to that of 1Ax1. Similar observations have been reported in transgenic wheat line B73-6-1 [23]. These might be caused by the insertion of foreign genes, with the genes silenced in the parents but activated in the cross progenies. However, the absence of some HMW-GS was the result of gene segregation in the cross progenies.
Four to five copies of the 1Ax1 gene have been observed as inserted into two loci in the chromosome of B102-1-2 [10,13,14], and we also found the 1Ax1 gene at two loci in the cross progeny. However, in contrast to 1Dx5 in cross progenies, the 1Ax1 gene in progenies of Chuan89-107�B102-1-2 expressed as an endogenetic gene. Its expression level was hardly changed and was approximately the same as that in the parent B102-1-2.
Our results indicated that the foreign genes could be inherited stably by their cross progenies. It was also suggested that the two foreign genes had different mechanisms of expression in the cross progeny, even though they were produced in the same way. The foreign 1Dx5 gene of 5-10 copies had the more complicated expression mechanism in our observation than the 1Ax1 gene of 4-5 copies.
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