Characterization of Agronomy, Grain Physicochemical Quality, and

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Characterization of agronomy, grain physicochemical quality and nutritional property of high-lysine 35R transgenic rice with simultaneous modification of lysine biosynthesis and catabolism Qingqing Yang, Hongyu Wu, Qianfeng Li, Ruxu Duan, Changquan Zhang, Samuel S. M. Sun, and Qiaoquan Liu J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 12 May 2017 Downloaded from http://pubs.acs.org on May 16, 2017

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Journal of Agricultural and Food Chemistry

Characterization of agronomy, grain physicochemical quality and nutritional property of high-lysine 35R transgenic rice with simultaneous modification of lysine biosynthesis and catabolism Qingqing Yang,

†,‡

†

Hongyu Wu, Qianfeng Li, *,‡,§

Samuel Saiming Sun, †

Qiaoquan Liu

†,§

†

Ruxu Duan, Changquan Zhang,

†,§

*,†,§

Key Laboratory of Crop Genetics and Physiology of Jiangsu Province / Key

Laboratory of Plant Functional Genomics of the Ministry of Education, College of Agriculture, Yangzhou University, Yangzhou 225009, China ‡

State Key Laboratory of Agrobiotechnology, School of Life Sciences, The Chinese

University of Hong Kong, Shatin, Hong Kong, China §

Co-Innovation Center for Modern Production Technology of Grain Crops of Jiangsu

Province / Joint International Research Laboratory of Agriculture and Agri-Product Safety of the Ministry of Education, Yangzhou University, Yangzhou 225009, China

*

Corresponding authors. Address (Q.Q.L.): College of Agriculture, Yangzhou

University, Yangzhou 225009, China. Tel.: +86 514 8797 9242. e-mail: [email protected]. Address (S.S.M.S.): School of Life Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong, China. Tel.: +852 260306337. e-mail: [email protected].

1

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ABBREVIATIONS USED

2

AAC, apparent amylose content; AC, amylose content; AK, aspartate kinase; BDV,

3

breakdown viscosity; CPV, cool paste viscosity; DHPS, dihydropicolinatesynthase;

4

DSC, diﬀerential scanning calorimeter; EAA, essential amino acid; GBSSI,

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granule-bound starch synthase I; GC, gel consistency; ∆H, enthalpy of gelatinization;

6

LKR/SDH,

7

dehydrogenase; PER, protein efficiency ratio; PeT, peak time; PaT, paste temperature;

8

PC, protein content; RVA, Rapid Visco Analyzer; Tc, conclusion temperature; To,

9

onset temperature; Tp, peak temperature; WT, wild type.

lysine

ketoglutaric

acid

reductase/saccharopine

dehydropine

10 11

ABSTRACT

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Lysine is the first limiting essential amino acid in rice. We previously constructed a

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series of transgenic rice lines to enhance lysine biosynthesis (35S), down-regulate its

14

catabolism (Ri), or simultaneously achieve both metabolic effects (35R). In this study,

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nine transgenic lines, three from each group, were selected for both field and animal

16

feeding trials. The results showed that the transgene(s) caused no obvious effects on

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field performance and main agronomic traits. Mature seeds of transgenic line 35R-17

18

contained 48-60-fold more free lysine than in wild type, and had slightly lower

19

apparent amylose content and softer gel consistency. Moreover, a 35-day feeding

20

experiment showed that the body weight gain, food efficiency, and protein efficiency

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ratio of rats fed the 35R-17 transgenic rice diet were improved when compared with

22

those fed wild type rice diet. These data will be useful for further evaluation and

23

potential commercialization of 35R high-lysine transgenic rice. 2


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KEYWORDS: Transgenic rice, Free lysine, Field performance, Feeding trial,

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Physicochemical quality

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INTRODUCTION

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Rice is one of the world’s most important grain crops and the main food for half the

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human population. Increasing the nutritional quality of rice has been the prior

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objective of rice breeders.1 The protein content and amino acid composition are the

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main indicators for evaluation of rice nutritional quality, especially the essential

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amino acid (EAA) content.2 In rice seeds, as well as in other cereal crops, lysine is the

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main limiting EAA, and lack of lysine can result in human metabolic disorders, even

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causing some physiological functional disorders.3 In addition, lack of dietary lysine

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can affect the absorption and utilization of proteins.4 Thus, there are many efforts to

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increase the lysine content in rice. Though several quantitative trait loci associated

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with lysine content were detected in rice5, and the mutants with high-lysine content

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were generated,6 the use of traditional plant breeding methods have enabled only

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limited progress in improving rice lysine content. It is particularly important to

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improve the grain lysine content in rice using genetic engineering.7-10 Recently,

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transgenic rice with increased lysine content were produced by over-accumulation of

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the proteins containing high proportion of lysine residues, such as endogenous

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histones, foreign lysine-rich protein from winged bean, but these high lysine

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transgenic rice usually displayed some negative effects on grain apparence.11, 12, 13

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Lysine is synthesized from the aspartic acid biosynthetic pathway in higher

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plants.4, 14, 15 In lysine biosynthesis, there are two key enzymes, aspartate kinase (AK)

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and dihydropicolinatesynthase (DHPS), and DHPS can be feedback inhibited by 3



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lysine.16-18 This lysine feedback inhibition of DHPS is less sensitive in bacteria, and

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the over-expression of bacterial DHPS in tobacco resulted in a 15-fold increase in the

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free lysine content compared with the wild type.16 Also, the level of free lysine has

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been significantly increased in transgenic Arabidopsis,19, 20 soybean,2 and tobacco16, 17

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by over-expression of the lysine feedback-insensitive bacterial DHPS gene. But,

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expression of feedback-insensitive lysine biosynthesis enzymes gave only a slight

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increase in free lysine levels in mature seeds of barley and rice.21, 22, as which is

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usually associated with an increase in lysine catabolism.2, 20, 22 This indicates that the

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accumulation and catabolism of lysine can be altered through its self-regulation,

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especially in seeds. Several studies have shown that inhibiting lysine catabolism by

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suppression of the LKR/SDH gene, which encodes lysine ketoglutaric acid

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reductase/saccharopine dehydropine dehydrogenase (LKR/SDH), resulted in a

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dramatic increase in the free lysine content of mature seeds in transgenic maize and

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rice.23-26

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The success of plant genetic engineering requires not only the ability to deliver

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functional DNA into the plant genome, but also stable inheritance, as well as the

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safety of the transgenic plant/food.27 Many studies have reported that the inheritance

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and stability of the transgenes varied in their offspring.28-33 Consequently, the field

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performance and nutritional evaluation of transgenic rice need to be determined,

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especially of the transgenic rice with output traits such as high lysine cereals. For

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example, early reports suggested that the high lysine sorghum mutant hl showed

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improved protein and nutritional quality in the grain.34 The starch granules were

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modified in high lysine barley and maize grains.35, 36 The total protein content in

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mature seeds of high lysine transgenic maize increased by 11.6% to 39.0% compared

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to the non-transgenic wild type.37 Out of 642 transgenic lysine-rich maize lines , the 4


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contents of total protein, lysine, some other amino acids, several minerals, and

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vitamin B2 were significantly higher than those in conventional Quality Protein

75

Maize.27

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In our previous study,25 in which the goal was to increase the lysine content of

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rice endosperm, we generated different types of transgenic rice lines (Table 1): (1)

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plants constitutively expressing the bacterial lysine feedback-insensitive AK and

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DHPS genes, both driven by the CaMV 35S promoter, called 35S; (2) transgenic

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plants in which the expression of the LKR/SDH gene is down-regulated by expression

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of an LKR-RNAi construct under the control of rice endosperm-specific glutelin Gt1

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promoter, called Ri; and (3) plants with combined expression of constructs (1) and (2),

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called 35R. The molecular characteristics and metabolite profiling of these different

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transgenic rice lines had been carefully studied, and we found that reduced LKR/SDH

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activity combined with over-expression of the bacterial AK and DHPS could

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dramatically increase the accumulation of free lysine in seeds of 35R transgenic rice.25

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However, the effects of the expression of these transgenes on plant growth, field

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performance, grain physicochemical quality, and nutritional value in these high-lysine

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transgenic rice lines are unknown. In this study, we selected nine transgenic rice lines

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carrying the above gene constructs for both field and feed trials, and here we describe

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the agronomy, grain physicochemical quality and nutritional property of these

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transgenic rice lines, especial of 35R lines, in detail.

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MATERIALS AND METHODS

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Plant Materials

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An elite rice cultivar, Oryza sativa ssp. Japonica cv. Wuxiangjing 9 (WXJ9), from 5



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China, and its derived nine transgenic rice lines were used in this study (Table 1).

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These nine transgenic lines were derived from three different DNA constructs: (1)

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35S-1, 35S-3, and 35S-13 showed constitutive expression of the bacterial lysine

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feedback-insensitive AK and DHPS genes, both driven by the CaMV 35S promoter; (2)

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Ri-17, Ri-18, and Ri-43 express an LKR-RNAi construct under the control of rice

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endosperm-specific glutelin Gt1 gene promoter to down regulate the LKR/SDH gene;

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and (3) 35R-17, 35R-56, and 35R-94 that express both constructs of (1) and (2).

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Production of the DNA constructs and molecular identification of transgenic rice lines

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were as described in Long et al.25 As described in our previous publication25, all

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selected nine transgenic lines were homozygous for the transgenes and in T4-T8

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generations for field trials in present study. The presence of the transgenes from three

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successive generations were further confirmed (Fig. S1), by PCR analysis with

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transgene-specific primers (Table S1) based on the methods of Long et al.25.

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Field Trials and Agronomic Trait Investigations

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The transgenic lines 35S, Ri, and 35R and the corresponding non-transgenic wild type

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(WT) were grown together from 2010 to 2015 in the experimental fields at Yangzhou

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University (Yangzhou, Jiangsu Province, China) under identical climatic conditions

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with permission for small-scale field trials from the Ministry of Agriculture, China.

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Three replicate plots were grown, and the tested lines were planted randomly in each

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plot. The main agronomic traits were recorded after maturity, and the mature seeds

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were harvested for grain quality analyses and feeding experiments.

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Grain Components and Quality Analyses

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The mature seeds were milled and processed into flour for analyses of grain

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components and physicochemical qualities.38 The total starch content, crude protein 6


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content, apparent amylase content (AAC), gel consistency (GC), and Rapid Visco

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Analyzer (RVA) profile were performed as previously described.38, 40 The contents of

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total and free amino acids were determined using the methods of Yang et al.26

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Starch Isolation and Characteristics

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Purified starch samples prepared from the milled rice endosperm using the neutral

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protease method as described previously.39 The amylose content and RVA profile of

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isolated starches were determined as above, and the starch thermal properties were

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measured with a differential scanning calorimeter (DSC 200 F3, Netzsch Instruments

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NA LLC; Burlington, MA) according to the methods of Zhang et al.39

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GBSSI Protein Analysis

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The granule-bound starch synthase I (GBSSI) was extracted from mature seeds as

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described by Liu et al.41 SDS-PAGE detection of GBSSI protein was performed using

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standard procedures, and gels were stained with Coomassie Brilliant Blue R250 to

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examine the protein bands.41

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Diet Preparation

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Four rice diets with high lysine levels were prepared based on the American Institute

137

of Nutrition AIN-93G formulation42 for feeding experiments (Table 2). Two diets,

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called Ri-17 and 35R-17, were prepared with rice flour from transgenic lines Ri-17

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and 35R, respectively, and the WT diet contained flour from the non-transgenic wild

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type WXJ9. A WT+Lys control diet was prepared from WT rice flour supplemented

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with crystalline L-lysine (Sigma Aldrich, USA), and the total lysine concentration was

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consistent with that of the 35R-17 diet (Table 2). All of the four diets contained the

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same levels of minerals, vitamins, and fiber, and the protein, amino acids, and starch

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were solely derived from rice and supplementary lysine, accounting for 85% (by 7



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weight) of the diets (Table 2). The rice diets were vacuum-packed in polyethylene

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bags until use.

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Animals and Feeding Trials

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Animal feeding experiments were carried out in an animal house of the Chinese

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University of Hong Kong (CUHK; Hong Kong, China). The experimental procedure

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was approved by the Animal Experimentation Ethics Committee of CUHK (AEEC No.

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14/131/MIS). All experiments were performed in accordance with the guidelines for

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the use of live animals. Male Sprague Dawley (SD) rats with initial body weights of

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70-80 g were used in the feeding trials. Initially, each rat received the same amount of

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diet, and, after two weeks, the rats had ad libitum access to feed and water. The body

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weight of each rat was measured every 2-3 days. Food intake (g diet/rat/day) was

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determined from the average amount of diet consumed by each rat over the course of

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35 d; food efficiency (%) was calculated as body weight gain (g)/food intake (g) ×

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100,43 and protein efficiency ratio (PER) was calculated as reported by Hoseini et al44:

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PER=(final body weight-initial body weight)/protein intake.

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Statistical Analyses

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Results are presented as mean ± SD. Statistical comparisons were designed to

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determine whether the differences between the transgenic and WT groups were

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attributed to lysine levels. Homogeneity of variance was determined by one-way

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analysis of variance (ANOVA) using SPSS 17.0 for windows (SPSS Inc., Chicago, IL,

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USA), followed by Tukey’s multiple-comparison test. Differences were considered

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statistically significant at P

Characterization of Agronomy, Grain Physicochemical Quality, and

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