通过代谢工程提高稻米游离赖氨酸含量的研究
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摘要
植物是人类和牲畜所消耗的蛋白质的主要来源,但其营养品质往往不够完全。一般来说,禾谷类作物种子蛋白质中的赖氨酸和色氨酸含量低。稻米所含能量高,其中的贮藏蛋白易被消化吸收,但其含量偏低,赖氨酸含量也较缺乏,是稻米蛋白质中的第一限制必需氨基酸。所以,提高稻米中的赖氨酸和蛋白质含量,平衡其营养品质,一直是遗传育种学家追求的目标之一。在对高等植物赖氨酸代谢过程认识的基础上,可设计对水稻种子中赖氨酸合成和降解途径分枝点上关键酶的表达进行调控研究,探求最大限度提高稻米中游离赖氨酸含量的可能途径;具体策略包括通过超表达对赖氨酸反馈抑制不敏感的赖氨酸合成酶关键基因AK(编码天冬氨酸激酶)和DHPS(编码二氢吡啶羧酸合酶),增加赖氨酸的合成;通过反义RNA或RNA干扰抑制水稻中分解赖氨酸关键酶基因LKR/SDH(编码赖氨酸酮戊二酸酯还原酶/酵母氨酸脱氢酶)的表达,减少水稻籽粒中赖氨酸的分解。本研究即是在本实验室已获得含赖氨酸代谢相关基因转基因水稻的基础上,分析比较不同转基因结构或组合对提高水稻籽粒中游离赖氨酸含量的效果,评价通过代谢工程提高赖氨酸含量后对稻米中其他氨基酸和品质性状以及植株生长等的影响。主要研究内容及结果如下:
1、为了解水稻LKR/SDH基因的表达调控,通过对GUS活性的定性与定量分析,比较研究了由水稻LKR/SDH基因不同长度启动子片段控制的GUS融合基因在转基因水稻植株不同组织中的表达。结果表明,翻译起始密码子ATG上游3.9kb的启动子控制的GUS基因在转基因水稻种子胚乳、叶和茎等多个组织中都能表达,但表现出一定的胚乳特异性。该基因5'端不同缺失长度启动子的表达特性不同,ATG上游2.9或2.0kb长启动子驱动GUS报告基因表达的能力要弱于3.9kb的,但1.6kb长启动子-GUS融合基因的表达活性最高,而且并不表现出组织特异性,说明在该基因ATG上游1.6至3.9kb区域内含有负责其精细表达的顺式调控元件。
2、在含不同赖氨酸代谢相关基因的转基因植株自交后代中,通过对目的基因的PCR和Southern杂交检测等技术,选育获得了较多的转基因纯合系和4个无潮霉素抗性选择标记基因的转基因新品系。Northern杂交表明,在水稻Gt1启动子或CaMV 35S启动子控制下,来源于大肠杆菌的AK和DHPS基因在大多数转基因水稻的发育种子能高效表达,导入LKR/SDH基因的RNAi结构后可明显抑制转基因水稻中内源LKR/SDH基因的表达。
3、测定并比较了各类转基因水稻成熟种子中的氨基酸含量,结果表明导入不同赖氨酸代谢相关基因及其组合对提高稻米游离赖氨酸含量的效果明显不同。只导入AK和DHPS基因的转基因稻米中游离赖氨酸含量变化不大;导入LKR/SDH-RNAi结构后稻米中游离赖氨酸含量可提高5-7倍;而同时导入3个基因的转基因稻米中游离赖氨酸含量提高幅度最大,最高的可比未转化对照高50倍。在游离赖氨酸含量提高幅度较大的转基因植株稻米中,总赖氨酸含量也有显著的提高,最高的可比未转化对照提高49%。
4、导入不同赖氨酸代谢相关基因后,对转基因稻米中的总氨基酸和总蛋白质含量没有产生显著的影响。但是,在游离赖氨酸含量提高幅度较大的转基因稻米中,其它游离氨基酸的含量也发生了一定的变化,表现为与赖氨酸合成相关的天冬氨酸、苏氨酸等的游离态含量有不同程度的下降。
5、与未转化受体亲本相比,大多数转入赖氨酸代谢相关基因的转基因植株成熟种子的外观品质、主要理化品质和田间农艺性状等并没有发生明显的变化。但是,在游离赖氨酸含量显著提高的部分转基因水稻中,其成熟籽粒的外观品质变差、直链淀粉含量降低、胶稠度变软、淀粉粘滞性谱变低,并对籽粒重也有一定的负效应。
Plants are the primary source of proteins consumed by humans and livestock. However, most plant proteins are nutritionally unbalanced, because they are deficient in certain essential amino acids. In general, cereal proteins are low in lysine and tryptophan. Rice (Oryza sativa L.), one of the leading food crops and the staple food of over half the world's population, is a very good and relatively cheap source of energy and protein. However, like other cereals, rice proteins are nutritionally incomplete due to their deficiency in threonine, tryptophan, especially lysine. Based on our understanding the lysine biosynthetic and catabolic pathway, two apporaches could be carried out to enhance free lysine content in rice. One is to increase the rate of lysine synthesis by bypassing the feedback regulation in lysine biosynthetic pathway by expressed the genes encoding E. coli feedback- insensitive aspartate kinase (AK) and dihydrodipicolinate synthase (DHPS). Another approach is to down-regulate the rate of lysine catabolism by designing antisense or RNAi constructs to inhibitate the expression of the LKR/SDH (lysine- ketoglutaric acid reductase and saccharopine dehydrogenase) bifunctional enzymes, which controlled the rate of lysine degradation.
In present study, by using the transgenic rice plants developed previously containing different target genes involved in lysine synthesis and catabolism described as above, the main purpose is to analysze and compare the effect of different target transgenes on enhancing lysine content in rce seeds, as well as to study the effect on rice growth, development and grain quality. The main results were showed as followings.
1. To understand the expression pattern of rice LKR/SDH gene the GUS activity was carefully measured and compared among different transgneic rice plants containing GUS fusion gene controlled by the promoters with different length of rice LKR / SDH gene. The results showed that the LKR/SDH promoter with a 3.9 kb length upstream of translation initiation codon ATG could drive the expression of GUS gene in endosperm, leaf and stem of transgenic rice plants, and prefer a certain endosperm-specific expression. It was less of GUS expression level when driven by the 2.9 or 2.0 kb promoter with 5 'deletion than that by the 3.9 kb one, but the expression of GUS fusion gene controlled by a short length promoter, 1.6 kb upstream of ATG, was detected to be highest, and not show tissue-specificity. Therefore, within the region of 1.6 to 3.9 kb upstream of ATG, there might contain several cis-regulatory elements responsible for the fine expression of rice LKR/SDH gene.
2. From the self-progeny of transgenic rice plants with different lysine metabolism-related genes, many homozygous transgenic lines were selected, and several transgenic lines without the hygromycin selectable marker gene were identified by both PCR ans Southern blot analyses. The results from Northern blots showed that the introduced AK and DHPS gene from E. coli, driven by either rice Gt1 or CaMV 35S promoter, could be highly expressed in developing seeds of most transgenic lines. In the endosperm of transgenic rice containing the LKR/SDH RNAi structure, the expression of endogenous LKR/SDH gene was significantly inhibited.
3. The amino acid content in mature seeds was determined and compared among various types of transgenic rice containing different lysine metabolism-related genes. The results showed that, after compared with that of untransformed wild type, there was no or little change of free lysine content in the seeds of transgenic rice carrying both AK and DHPS genes. In the seeds of LKR/SDH-RNAi transgenic rice plants, the free lysine content was detected to increase by 5-7 times. After combined the three transgenes (AK, DHPS and LKR/SDH-RNAi) into the same rice plant, free lysine content in seeds siginificantly increased, and the maximum increase level was about 50 times over that of wild type. In transgenic lines with highly increased free lysine, the total lysine content was also significantly enhanced, the highest was about 49 percentage over that of wild type.
4. The data of total protein and amino acid analyses revealed that there was no or very little effect of the introduced target genes on total seed protein and total amino acid contents in transgenic rice. However, in transgenic rice with significantly increased free lysine level, some other free amino acids’content were also changed, especially aimino acids related to lysine metabolism pathway, such as threonine and aspartic acid.
5. The results from field trials and quality analyses showed that there was no or limited effect of the transgenes on main agronomic traits and grain quality in most of the transgneic rice lines. But, in transgenic rice with significantly increased free lysine level, the grains showed more chalkiness, lower amylose content, softer gel consistency, and little lower starch viscosity when compared with those of wild type.
1、为了解水稻LKR/SDH基因的表达调控,通过对GUS活性的定性与定量分析,比较研究了由水稻LKR/SDH基因不同长度启动子片段控制的GUS融合基因在转基因水稻植株不同组织中的表达。结果表明,翻译起始密码子ATG上游3.9kb的启动子控制的GUS基因在转基因水稻种子胚乳、叶和茎等多个组织中都能表达,但表现出一定的胚乳特异性。该基因5'端不同缺失长度启动子的表达特性不同,ATG上游2.9或2.0kb长启动子驱动GUS报告基因表达的能力要弱于3.9kb的,但1.6kb长启动子-GUS融合基因的表达活性最高,而且并不表现出组织特异性,说明在该基因ATG上游1.6至3.9kb区域内含有负责其精细表达的顺式调控元件。
2、在含不同赖氨酸代谢相关基因的转基因植株自交后代中,通过对目的基因的PCR和Southern杂交检测等技术,选育获得了较多的转基因纯合系和4个无潮霉素抗性选择标记基因的转基因新品系。Northern杂交表明,在水稻Gt1启动子或CaMV 35S启动子控制下,来源于大肠杆菌的AK和DHPS基因在大多数转基因水稻的发育种子能高效表达,导入LKR/SDH基因的RNAi结构后可明显抑制转基因水稻中内源LKR/SDH基因的表达。
3、测定并比较了各类转基因水稻成熟种子中的氨基酸含量,结果表明导入不同赖氨酸代谢相关基因及其组合对提高稻米游离赖氨酸含量的效果明显不同。只导入AK和DHPS基因的转基因稻米中游离赖氨酸含量变化不大;导入LKR/SDH-RNAi结构后稻米中游离赖氨酸含量可提高5-7倍;而同时导入3个基因的转基因稻米中游离赖氨酸含量提高幅度最大,最高的可比未转化对照高50倍。在游离赖氨酸含量提高幅度较大的转基因植株稻米中,总赖氨酸含量也有显著的提高,最高的可比未转化对照提高49%。
4、导入不同赖氨酸代谢相关基因后,对转基因稻米中的总氨基酸和总蛋白质含量没有产生显著的影响。但是,在游离赖氨酸含量提高幅度较大的转基因稻米中,其它游离氨基酸的含量也发生了一定的变化,表现为与赖氨酸合成相关的天冬氨酸、苏氨酸等的游离态含量有不同程度的下降。
5、与未转化受体亲本相比,大多数转入赖氨酸代谢相关基因的转基因植株成熟种子的外观品质、主要理化品质和田间农艺性状等并没有发生明显的变化。但是,在游离赖氨酸含量显著提高的部分转基因水稻中,其成熟籽粒的外观品质变差、直链淀粉含量降低、胶稠度变软、淀粉粘滞性谱变低,并对籽粒重也有一定的负效应。
Plants are the primary source of proteins consumed by humans and livestock. However, most plant proteins are nutritionally unbalanced, because they are deficient in certain essential amino acids. In general, cereal proteins are low in lysine and tryptophan. Rice (Oryza sativa L.), one of the leading food crops and the staple food of over half the world's population, is a very good and relatively cheap source of energy and protein. However, like other cereals, rice proteins are nutritionally incomplete due to their deficiency in threonine, tryptophan, especially lysine. Based on our understanding the lysine biosynthetic and catabolic pathway, two apporaches could be carried out to enhance free lysine content in rice. One is to increase the rate of lysine synthesis by bypassing the feedback regulation in lysine biosynthetic pathway by expressed the genes encoding E. coli feedback- insensitive aspartate kinase (AK) and dihydrodipicolinate synthase (DHPS). Another approach is to down-regulate the rate of lysine catabolism by designing antisense or RNAi constructs to inhibitate the expression of the LKR/SDH (lysine- ketoglutaric acid reductase and saccharopine dehydrogenase) bifunctional enzymes, which controlled the rate of lysine degradation.
In present study, by using the transgenic rice plants developed previously containing different target genes involved in lysine synthesis and catabolism described as above, the main purpose is to analysze and compare the effect of different target transgenes on enhancing lysine content in rce seeds, as well as to study the effect on rice growth, development and grain quality. The main results were showed as followings.
1. To understand the expression pattern of rice LKR/SDH gene the GUS activity was carefully measured and compared among different transgneic rice plants containing GUS fusion gene controlled by the promoters with different length of rice LKR / SDH gene. The results showed that the LKR/SDH promoter with a 3.9 kb length upstream of translation initiation codon ATG could drive the expression of GUS gene in endosperm, leaf and stem of transgenic rice plants, and prefer a certain endosperm-specific expression. It was less of GUS expression level when driven by the 2.9 or 2.0 kb promoter with 5 'deletion than that by the 3.9 kb one, but the expression of GUS fusion gene controlled by a short length promoter, 1.6 kb upstream of ATG, was detected to be highest, and not show tissue-specificity. Therefore, within the region of 1.6 to 3.9 kb upstream of ATG, there might contain several cis-regulatory elements responsible for the fine expression of rice LKR/SDH gene.
2. From the self-progeny of transgenic rice plants with different lysine metabolism-related genes, many homozygous transgenic lines were selected, and several transgenic lines without the hygromycin selectable marker gene were identified by both PCR ans Southern blot analyses. The results from Northern blots showed that the introduced AK and DHPS gene from E. coli, driven by either rice Gt1 or CaMV 35S promoter, could be highly expressed in developing seeds of most transgenic lines. In the endosperm of transgenic rice containing the LKR/SDH RNAi structure, the expression of endogenous LKR/SDH gene was significantly inhibited.
3. The amino acid content in mature seeds was determined and compared among various types of transgenic rice containing different lysine metabolism-related genes. The results showed that, after compared with that of untransformed wild type, there was no or little change of free lysine content in the seeds of transgenic rice carrying both AK and DHPS genes. In the seeds of LKR/SDH-RNAi transgenic rice plants, the free lysine content was detected to increase by 5-7 times. After combined the three transgenes (AK, DHPS and LKR/SDH-RNAi) into the same rice plant, free lysine content in seeds siginificantly increased, and the maximum increase level was about 50 times over that of wild type. In transgenic lines with highly increased free lysine, the total lysine content was also significantly enhanced, the highest was about 49 percentage over that of wild type.
4. The data of total protein and amino acid analyses revealed that there was no or very little effect of the introduced target genes on total seed protein and total amino acid contents in transgenic rice. However, in transgenic rice with significantly increased free lysine level, some other free amino acids’content were also changed, especially aimino acids related to lysine metabolism pathway, such as threonine and aspartic acid.
5. The results from field trials and quality analyses showed that there was no or limited effect of the transgenes on main agronomic traits and grain quality in most of the transgneic rice lines. But, in transgenic rice with significantly increased free lysine level, the grains showed more chalkiness, lower amylose content, softer gel consistency, and little lower starch viscosity when compared with those of wild type.
引文
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Galili, S., Guenoune, D., Wininger, S., Hana, B., Schupper, A., Ben-Dor, B. and
Kapulnik, Y. (2000) Enhanced levels of free and protein-bound threonine in transgenic alfalfa (Medicago sativa L.) expressing a bacterial feedback-insensitive aspartate kinase gene. Transgenic Res. 9, 137-144.
Gaziola, S. A., Teixeira, C. M. G., Lugli, J., Sodek, L. and Azevedo, R. A. (1997) The enzymology of lysine catabolism in rice seeds - Isolation, characterization, and regulatory properties of a lysine 2-oxoglutarate reductase saccharopine dehydrogenase bifunctional polypeptide. European Journal of Biochemistry 247, 364-371.
Gaziola, S. A., Sodek, L., Arruda, P., Lea, P. J. and Azevedo, R. A. (2000) Degradation of lysine in rice seeds: Effect of calcium, ionic strength, S-adenosylmethionine and S-2-aminoethyl-L-cysteine on the lysine 2-oxoglutarate reductase-saccharopine dehydrogenase bifunctional enzyme. Physiol. Plantarum 110, 164-171.
George AA and de Lumen BO (1991). A novel methionine-rich protein in soybean seed:identification, amino acid composition, and N-terminal sequence. J. Agric. Food Chem. 39: 224- 227.
GoncalvesButruille, M., Szajner, P., Torigoi, E., Leite, A. and Arruda, P. (1996) Purification and characterization of the bifunctional enzyme lysine-ketoglutarate reductase-saccharopine dehydrogenase from maize. Plant Physiol. 110, 765-771.
Green, C. E. and Phillips, R. L. (1974) Potential selection system for mutants with increased lysine, threonine, and methionine in cereal crops. Crop Sci. 14, 827-830.
Habben JE, Kiriels AW and Larkins BA (1993). The origin of lysine-containing proteins in opaque-2 maize endosperm. Plant Mol. Biol. 23: 825-838.
Habben JE and Larkins BA (1995). Genetic modification of seed proteins. Current Opinion in Biotechnology 6: 171一174.
Hoffman LM, Donaldson DD, Bookland R, Rashka K and Herman EM (1987).Synthesis and protein body deposition of maize 15-kDa zein in transgenic tobacco seeds. EMBO J. 6: 3213-3221.
Jacobsen, E. (1986) Isolation, Characterization and regeneration of an S-(2-aminoethyl)L-cysteine resistant cell-line of dihaploid potato. J. Plant Physiol. 123, 307-315.
Jefferson EA.(1987) Assaying Chimeric genes in Plants:The GUS gene fusion systrm Plant.Mol.Biol.Rep.5:387-405
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Kaneko, T., Hashimoto, T., Kumpaisal, R. and Yamada, Y. (1990) Molecular-cloning of wheat dihydrodipicolinate synthase. J. Biol. Chem. 265, 17451-17455.
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Karchi, H., Shaul, O. and Galili, G. (1994) Seed-specific expression of a bacterial desensitized aspartate kinase increases the production of seed threonine and methionine in transgenic tobacco. Plant Journal 3, 721-727.
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Galili, G., Tang, G. L., Zhu, X. H., Karchi, H., Miron, D., Gakiere, B. and Stepansky, A. (2001b) Molecular genetic dissection and potential manipulation of lysine metabolism in seeds. J. Plant Physiol. 158, 515-520.
Galili, S., Guenoune, D., Wininger, S., Hana, B., Schupper, A., Ben-Dor, B. and
Kapulnik, Y. (2000) Enhanced levels of free and protein-bound threonine in transgenic alfalfa (Medicago sativa L.) expressing a bacterial feedback-insensitive aspartate kinase gene. Transgenic Res. 9, 137-144.
Gaziola, S. A., Teixeira, C. M. G., Lugli, J., Sodek, L. and Azevedo, R. A. (1997) The enzymology of lysine catabolism in rice seeds - Isolation, characterization, and regulatory properties of a lysine 2-oxoglutarate reductase saccharopine dehydrogenase bifunctional polypeptide. European Journal of Biochemistry 247, 364-371.
Gaziola, S. A., Sodek, L., Arruda, P., Lea, P. J. and Azevedo, R. A. (2000) Degradation of lysine in rice seeds: Effect of calcium, ionic strength, S-adenosylmethionine and S-2-aminoethyl-L-cysteine on the lysine 2-oxoglutarate reductase-saccharopine dehydrogenase bifunctional enzyme. Physiol. Plantarum 110, 164-171.
George AA and de Lumen BO (1991). A novel methionine-rich protein in soybean seed:identification, amino acid composition, and N-terminal sequence. J. Agric. Food Chem. 39: 224- 227.
GoncalvesButruille, M., Szajner, P., Torigoi, E., Leite, A. and Arruda, P. (1996) Purification and characterization of the bifunctional enzyme lysine-ketoglutarate reductase-saccharopine dehydrogenase from maize. Plant Physiol. 110, 765-771.
Green, C. E. and Phillips, R. L. (1974) Potential selection system for mutants with increased lysine, threonine, and methionine in cereal crops. Crop Sci. 14, 827-830.
Habben JE, Kiriels AW and Larkins BA (1993). The origin of lysine-containing proteins in opaque-2 maize endosperm. Plant Mol. Biol. 23: 825-838.
Habben JE and Larkins BA (1995). Genetic modification of seed proteins. Current Opinion in Biotechnology 6: 171一174.
Hoffman LM, Donaldson DD, Bookland R, Rashka K and Herman EM (1987).Synthesis and protein body deposition of maize 15-kDa zein in transgenic tobacco seeds. EMBO J. 6: 3213-3221.
Jacobsen, E. (1986) Isolation, Characterization and regeneration of an S-(2-aminoethyl)L-cysteine resistant cell-line of dihaploid potato. J. Plant Physiol. 123, 307-315.
Jefferson EA.(1987) Assaying Chimeric genes in Plants:The GUS gene fusion systrm Plant.Mol.Biol.Rep.5:387-405
Juliano B O, Gu C.(1995)Rice and Human Nutrition.Beijing: China Agricultural University Press.31-4.
Kaneko, T., Hashimoto, T., Kumpaisal, R. and Yamada, Y. (1990) Molecular-cloning of wheat dihydrodipicolinate synthase. J. Biol. Chem. 265, 17451-17455.
Karchi, H., Miron, D., Benyaacov, S. and Galili, G. (1995) The lysine-dependent stimulation of lysine catabolism in tobacco seed requires calcium and protein-phosphorylation. Plant Cell 7, 1963-1970.
Karchi, H., Shaul, O. and Galili, G. (1994) Seed-specific expression of a bacterial desensitized aspartate kinase increases the production of seed threonine and methionine in transgenic tobacco. Plant Journal 3, 721-727.
Keeler SJ, Maloney CL, Webber PY, Patterson C, Hirata LT, Falco SC and Rice JA (1997).Expression of de novo high-lysine a-helical coiled-coil proteins may significantly increase the accumulated levels of lysine in mature seeds of transgenic tobacco plants.Plant Mol. Biol. 34:15-29.
Kemper, E. L., Neto, G. C., Papes, F., Moraes, K. C. M., Leite, A. and Arruda, P. (1999) The role of opaque-2 in the control of lysine-degrading activities in developing maize endosperm. Plant Cell 11, 1981-1993.
Kho CJ and de Lumen BO (1988). Identification and isolation of methionine-cysteine rich proteins in soybean seed: Plant Foods&Human Nutrition 38: 287-296.
Lea, P. J., Blackwell, R. D. and Azevedo, R. A. (1992) Analysis of barley metabolism using mutant genes; in Barley: genetics, biochemistry, molecular biology and biotechnology, Shewry, P. R. (ed.), pp. 181-208, CAB International, Wallingford.
Lea, P. J. and Ireland, R. J. (1999) Nitrogen metabolism in higher plants; in Plant amino acids: biochemistry and biotechnology, Singh, B. K. (ed.), pp. 1-48, Marcel Dekker, New York.
Lee, L. K. and Roth, C. M. (2003) Antisense technology in molecular and cellular bioengineering. Curr. Opin. Biotechnol. 14, 505-511.
Lee, S. I., Kim, H. U., Lee, Y. H., Suh, S. C., Lim, Y. P., Lee, H. Y. and Kim, H. I. (2001) Constitutive and seed-specific expression of a maize lysine-feedback- insensitive dihydrodipicolinate synthase gene leads to increased free lysine levels in rice seeds. Mol. Breed. 8, 75-84.
Matthews, B. F. (1999) Lysine, threonine and methionine biosynthesis; in Plant amino acids: biochemistry and biotechnology, Singh, B. K. (ed.), pp. 205-225, Marcel Dekker, New York.
Mattews BF and Hughes CA (1993). Nutritional improvement of the aspartate family of amino acids in edible crop plants. Amino acids 4: 21-34.
Mazur, B., Krebbers, E. and Tingey, S. (1999) Gene discovery and product development for grain quality traits. Science 285, 372-375.
Merlo, A. O., Cowen, N., Delate, T., Edington, B., Folkerts, O., Hopkins, N., Lemeiux, C., Skokut, T., Smith, K., Woosley, A., Yang, Y. J., Young, S. and Zwick, M. (1998) Ribozymes targeted to stearoyl-ACPΔ9 desaturase mRNA produce heritable increases of stearic acid in transgenic maize leaves. Plant Cell 10, 1603-1621.
Mertz, E. T., Bates, L. S. and Nelson, O. E. (1964) Mutant gene that changes protein composition and increase lysine content of maize endosperm. Science 145, 279-280.
Muehlbauer, G. J., Somers, D. A., Matthews, B. F. and Gengenbach, B. G. (1994) Molecular-genetics of the maize (Zea mays L.) aspartate kinase homoserine dehydrogenase gene family. Plant Physiol. 106, 1303-1312.
Munck, L., Karlsson, K. E., Hagberg, A. and Eggum, B. O. (1970) Gene for improved nutritional value in barley seed protein. Science 168, 985-&.
Murray, M. G. and Thompson, W. F. (1980) Rapid isolation of high molecular weight plant DNA. Nucleic Acid Res.8: 4321-4325
Ohtani T, Gahli G, Wallace, J.C., Thompson, G.A., Larkins, B.A. (1991). Normal and lysine-containing zeins are unstable in transgenic tobacco seeds. Plant Mol. Biol. 16: 117-128.
Shaul, O. and Galili, G. (1992) Increased lysine synthesis in tobacco plants that express high-levels of bacterial dihydrodipicolinate synthase in their chloroplasts. Plant Journal 2, 203-209.
Shihshieh Huang, Diane E. Kruger, Alessandra Frizzi.(2005). High-lysine corn produced by the combination ofenhanced lysine biosynthesis and reduced zein accumulation. Plant Biotechnology Journal.3:1-15
Singh, R. and Axtell, J. D. (1973) High lysine mutant gene (Hl) that improves protein quality and biological value of grain-sorghum. Crop Sci. 13, 535-539.
Sodek, L. and Wilson, C. M. (1970) Incorporation of leucine-C14 and lysine-C14 into protein in developing endosperm of normal and opaque-2 corn. Arch. Biochem. Biophys. 140, 29-30.
Sun SSM, Slightom, J.L. and Hall, T.C. (1987). Intervening sequences in a plant gene-comparison of the partial sequence of cDNA and genomic DNA of French bean phaseolin .Nature 289: 37-41.
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